Image differentiated multiplex assays

ABSTRACT

Provided herein are encoded microcarriers for analyte detection in multiplex assays. The microcarriers are encoded with an analog code for identification and include a capture agent for analyte detection. Also provided are methods of making the encoded microcarriers disclosed herein. Further provided are methods and kits for conducting a multiplex assay using the microcarriers described herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/374,930, filed Dec. 9, 2016, which is a continuation of InternationalPatent Application No. PCT/IB2016/000937, filed internationally on Jun.10, 2016 which claims the priority benefit of U.S. ProvisionalApplication Ser. No. 62/174,401, filed Jun. 11, 2015, the disclosures ofeach of which are incorporated herein by reference in their entirety.

FIELD

Provided herein are encoded microcarriers for analyte detection inmultiplex assays, as well as methods of making and using the same andkits related thereto. The microcarriers are encoded with an analog codefor identification and include a capture agent for capturing an analyte.

BACKGROUND

Immunological and molecular diagnostic assays play a critical role bothin the research and clinical fields. Often it is necessary to performassays for a panel of multiple targets to gain a meaningful orbird's-eye view of results to facilitate research or clinicaldecision-making. This is particularly true in the era of genomics andproteomics, where an abundance of genetic markers and/or biomarkers arethought to influence or be predictive of particular disease states. Intheory, assays of multiple targets can be accomplished by testing eachtarget separately in parallel or sequentially in different reactionvessels (i.e., multiple singleplexing). However, not only are assaysadopting a singleplexing strategy often cumbersome, but they alsotypically required large sample volumes, especially when the targets tobe analyzed are large in number.

A multiplex assay simultaneously measures multiple analytes (two ormore) in a single assay. Multiplex assays are commonly used inhigh-throughput screening settings, where many specimens can be analyzedat once. It is the ability to assay many analytes simultaneously andmany specimens in parallel that is the hallmark of multiplex assays andis the reason that such assays have become a powerful tool in fieldsranging from drug discovery to functional genomics to clinicaldiagnostics. In contrast to singleplexing, by combining all targets inthe same reaction vessel, the assay is much less cumbersome and mucheasier to perform, since only one reaction vessel is handled per sample.The required test samples can thus be dramatically reduced in volume,which is especially important when samples (e.g., tumor tissues,cerebral spinal fluid, or bone marrow) are difficult and/or invasive toretrieve in large quantities. Equally important is the fact that thereagent cost can be decreased and assay throughput increaseddrastically.

Many assays of complex macromolecule samples are composed of two steps.In the first step, agents capable of specifically capturing the targetmacromolecules are attached to a solid phase surface. These immobilizedmolecules may be used to capture the target macromolecules from acomplex sample by various means, such as hybridization (e.g., in DNA,RNA based assays) or antigen-antibody interactions (in immunoassays). Inthe second step, detection molecules are incubated with and bind to thecomplex of capture molecule and the target, emitting signals such asfluorescence or other electromagnetic signals. The amount of the targetis then quantified by the intensity of those signals.

Multiplex assays may be carried out by utilizing multiple captureagents, each specific for a different target macromolecule. Inchip-based array multiplex assays, each type of capture agent isattached to a pre-defined position on the chip. The amount of multiplextargets in a complex sample is determined by measuring the signal of thedetection molecule at each position corresponding to a type of captureagent. In suspension array multiplex assays, microparticles ormicrocarriers are suspended in the assay solution. These microparticlesor microcarriers contain an identification element, which may beembedded, printed, or otherwise generated by one or more elements of themicroparticle/microcarrier. Each type of capture agent is immobilized toparticles with the same ID, and the signals emitted from the detectionmolecules on the surface of the particles with a particular ID reflectthe amount of the corresponding target.

Existing systems for suspension array multiplex assays are limited inresolution. Some multiplex systems use digital barcodes printed on flatmicrobeads using standard semiconductor fabrication techniques. However,the number of identifiers that can be generated by a particular numberof digits is limited. Increasing the number of unique identifiersrequires increasing the number of barcode digits, thus requiring morespace for printing on an already tiny microbead. Another type ofmultiplex system uses color-coding, such as fluorescent beads encodedwith a unique fluorescent dye. However, the number of unique identifiersavailable for such fluorescent systems is limited due to overlappingexcitation/emission spectra, and identification errors may arise from,e.g., batch-to-batch variation in fluorescent dyes.

Therefore, a need exists for an analog-encoded multiplex assay system,e.g., one not constrained by limitations such as digital barcode size orfluorophore resolution. Such a system allows nearly unlimited uniqueidentifiers and minimizes recognition error due to the use of analogcodes (e.g., from overlapping spectra or batch-to-batch fluorophorevariation).

All publications, patents, and patent applications cited herein arehereby incorporated by reference in their entirety for all purposes.

BRIEF SUMMARY

To meet this need, provided herein, inter alia, are microcarriers,encoded with an analog code, that include a capture agent for capturingan analyte. These microcarriers may be used, for example, in multiplexedassays in which each microcarrier includes a capture agent for capturinga specific analyte and an analog code for identification. Methods ofmaking and using such microcarriers, as well as kits related thereto,are further provided.

Accordingly, in one aspect, provided herein is an encoded microcarriercomprising (a) a substantially transparent polymer layer having a firstsurface and a second surface, the first and the second surfaces beingparallel to each other; (b) a substantially non-transparent polymerlayer, wherein the substantially non-transparent polymer layer isaffixed to the first surface of the substantially transparent polymerlayer and encloses a center portion of the substantially transparentpolymer layer, and wherein the substantially non-transparent polymerlayer comprises a two-dimensional shape representing an analog code; and(c) a capture agent for capturing an analyte, wherein the capture agentis coupled to at least one of the first surface and the second surfaceof the substantially transparent polymer layer in at least the centerportion of the substantially transparent polymer layer.

In some embodiments, the microcarrier further comprises (d) a magnetic,substantially non-transparent layer that encloses the center portion ofthe substantially transparent polymer layer between the substantiallynon-transparent polymer layer and the center portion of thesubstantially transparent polymer layer, wherein the magnetic,substantially non-transparent layer is affixed to the first surface orthe second surface of the substantially transparent polymer layer. Insome embodiments, the microcarrier further comprises (e) a secondsubstantially transparent polymer layer aligned with the firstsubstantially transparent polymer layer, the second substantiallytransparent polymer layer having a center portion that is aligned withthe center portion of the first substantially transparent polymer layer,wherein the second substantially transparent polymer layer is affixed tothe second surface of the first substantially transparent polymer layerand does not extend beyond the two-dimensional shape of the firstsubstantially transparent polymer layer; and (f) a magnetic,substantially non-transparent layer that encloses the center portion ofthe first substantially transparent polymer layer between thesubstantially non-transparent polymer layer and the center portion ofthe substantially transparent polymer layer, wherein the magnetic,substantially non-transparent layer is affixed between the first and thesecond substantially transparent polymer layers. In some embodiments,the microcarrier further comprises an orientation indicator fororienting the analog code of the substantially non-transparent polymerlayer. In some embodiments, the orientation indicator comprises anasymmetry of the magnetic, substantially non-transparent layer. In someembodiments, the magnetic, substantially non-transparent layer comprisesnickel. In some embodiments, the magnetic, substantially non-transparentlayer is between about 50 nm and about 10 μm in thickness. In someembodiments, the magnetic, substantially non-transparent layer is about0.1 μm in thickness. In some embodiments, the two-dimensional shape ofthe substantially non-transparent polymer layer comprises a gear shapecomprising a plurality of gear teeth, and wherein the analog code isrepresented by one or more aspects selected from the group consisting ofthe height of one or more gear teeth of the plurality, the width of oneor more gear teeth of the plurality, the number of gear teeth in theplurality, and the arrangement of one or more gear teeth within theplurality. In some embodiments, the plurality of gear teeth comprisesone or more gear teeth that are between about 1 μm and about 10 μm wide.In some embodiments, the plurality of gear teeth comprises one or moregear teeth that are between about 1 μm and about 10 μm tall. In someembodiments, the plurality of gear teeth comprises two or more gearteeth that are spaced between about 1 μm and about 10 μm apart. In someembodiments, the microcarrier further comprises (g) one or more columnsprojecting from the first surface of the first substantially transparentpolymer layer, wherein the one or more columns are not within the centerportion of the first substantially transparent polymer layer; and/or (h)one or more columns projecting from the second surface of the firstsubstantially transparent polymer layer or a surface of the secondsubstantially transparent polymer layer that is not affixed to the firstsubstantially transparent polymer layer, wherein the one or more columnsare not within the center portions of the first or the secondsubstantially transparent polymer layer. In some embodiments, themicrocarrier is a substantially circular disc. In some embodiments, thecenter portion of the first substantially transparent polymer layercomprises between about 5% and about 90% of the surface area of thefirst substantially transparent polymer layer. In some embodiments, thecenter portion of the first substantially transparent polymer layercomprises about 25% of the surface area of the first substantiallytransparent polymer layer. In some embodiments, the microcarrier is lessthan about 200 μm in diameter. In some embodiments, the microcarrier isabout 50 μm in diameter. In some embodiments, the microcarrier is lessthan about 50 μm in thickness. In some embodiments, the microcarrier isabout 10 μm in thickness. In some embodiments, the analyte is selectedfrom the group consisting of a DNA molecule, a DNA-analog-molecule, anRNA-molecule, an RNA-analog-molecule, a polynucleotide, a protein, anenzyme, a lipid, a phospholipid, a carbohydrate moiety, apolysaccharide, an antigen, a virus, a cell, an antibody, a smallmolecule, a bacterial cell, a cellular organelle, and an antibodyfragment. In some embodiments, the capture agent for capturing theanalyte is selected from the group consisting of a DNA molecule, aDNA-analog-molecule, an RNA-molecule, an RNA-analog-molecule, apolynucleotide, a protein, an enzyme, a lipid, a phospholipid, acarbohydrate moiety, a polysaccharide, an antigen, a virus, a cell, anantibody, a small molecule, a bacterial cell, a cellular organelle, andan antibody fragment. In some embodiments, the substantially transparentpolymer of the first or the second substantially transparent polymerlayer comprises an epoxy-based polymer. In some embodiments, theepoxy-based polymer is SU-8.

In another aspect, provided herein is an encoded microcarrier comprising(a) a substantially non-transparent polymer layer having a first surfaceand a second surface, the first and the second surfaces being parallelto each other, wherein an outline of the substantially non-transparentpolymer layer comprises a two-dimensional shape that represents ananalog code; and (b) a capture agent for capturing an analyte, whereinthe capture agent is coupled to at least one of the first surface andthe second surface of the substantially non-transparent polymer layer inat least a center portion of the substantially non-transparent polymerlayer.

In some embodiments, the microcarrier further comprises (c) one or morecolumns projecting from the first surface and/or the second surface ofthe substantially non-transparent polymer layer, wherein the one or morecolumns comprise a magnetic material. In some embodiments, the one ormore columns are between about 1 μm and about 10 μm tall. In someembodiments, the one or more columns are between about 1 μm and about 10μm in diameter. In some embodiments, the microcarrier further comprises(d) a magnetic layer comprising a magnetic material affixed to thesecond surface of the substantially non-transparent polymer layer,wherein the magnetic layer does not extend beyond the center portion ofthe substantially non-transparent polymer layer, and wherein the captureagent is coupled to at least the first surface of the substantiallynon-transparent polymer layer. In some embodiments, the microcarrierfurther comprises (e) a second substantially non-transparent polymerlayer aligned with the first substantially non-transparent polymerlayer, wherein the second substantially non-transparent polymer layer isaffixed to the second surface of the first substantially transparentpolymer layer and does not extend beyond the outline of the firstsubstantially transparent polymer layer, and wherein the magnetic layeris affixed between the first and the second substantially transparentpolymer layers. In some embodiments, the magnetic material comprisesnickel. In some embodiments, the microcarrier further comprises anorientation indicator for orienting the analog code of the substantiallynon-transparent polymer layer. In some embodiments, the orientationindicator comprises an asymmetry of the outline of the substantiallynon-transparent polymer layer. In some embodiments, the outline of thesubstantially non-transparent polymer layer comprises a two-dimensionalgear shape comprising a plurality of gear teeth, and wherein the analogcode is represented by one or more aspects selected from the groupconsisting of the height of one or more gear teeth of the plurality, thewidth of one or more gear teeth of the plurality, the number of gearteeth in the plurality, and the arrangement of one or more gear teethwithin the plurality. In some embodiments, the plurality of gear teethcomprises one or more gear teeth that are between about 1 μm and about10 μm wide. In some embodiments, the plurality of gear teeth comprisesone or more gear teeth that are between about 1 μm and about 10 μm tall.In some embodiments, the plurality of gear teeth comprises two or moregear teeth that are spaced between about 1 μm and about 10 μm apart. Insome embodiments, the microcarrier is a substantially circular disc. Insome embodiments, the center portion of the first substantiallynon-transparent polymer layer comprises between about 5% and about 90%of the surface area of the first substantially non-transparent polymerlayer. In some embodiments, the center portion of the firstsubstantially non-transparent polymer layer comprises about 25% of thesurface area of the first substantially non-transparent polymer layer.In some embodiments, the microcarrier is less than about 200 μm indiameter. In some embodiments, the microcarrier is about 60 μm indiameter. In some embodiments, the microcarrier is less than about 50 μmin thickness. In some embodiments, the microcarrier is about 10 μm inthickness. In some embodiments, the analyte is selected from the groupconsisting of a DNA molecule, a DNA-analog-molecule, an RNA-molecule, anRNA-analog-molecule, a polynucleotide, a protein, an enzyme, a lipid, aphospholipid, a carbohydrate moiety, a polysaccharide, an antigen, avirus, a cell, an antibody, a small molecule, a bacterial cell, acellular organelle, and an antibody fragment. In some embodiments, thecapture agent for capturing the analyte is selected from the groupconsisting of a DNA molecule, a DNA-analog-molecule, an RNA-molecule, anRNA-analog-molecule, a polynucleotide, a protein, an enzyme, a lipid, aphospholipid, a carbohydrate moiety, a polysaccharide, an antigen, avirus, a cell, an antibody, a small molecule, a bacterial cell, acellular organelle, and an antibody fragment. In some embodiments, thesubstantially non-transparent polymer comprises an epoxy-based polymer.In some embodiments, the substantially non-transparent polymer comprisesa black matrix resist.

In another aspect, provided herein is a method of making an encodedmicrocarrier, comprising: (a) depositing a substantially transparentpolymer layer, wherein the substantially transparent polymer layer has afirst surface and a second surface, the first and the second surfacesbeing parallel to each other; (b) depositing a magnetic, substantiallynon-transparent layer on the first surface of the substantiallytransparent polymer layer; (c) etching the magnetic, substantiallynon-transparent layer to remove a portion of the magnetic, substantiallynon-transparent layer that is deposited over a center portion of thesubstantially transparent polymer layer; (d) depositing a secondsubstantially transparent polymer layer over the magnetic, substantiallynon-transparent layer, wherein the second substantially transparentpolymer layer has a first surface and a second surface, the first andthe second surfaces being parallel to each other, wherein the secondsurface is affixed to the magnetic, substantially non-transparent layer,and wherein the second substantially transparent polymer layer isaligned with the first substantially transparent polymer layer and has acenter portion that is aligned with the center portion of thesubstantially transparent polymer layer; and (e) depositing asubstantially non-transparent polymer layer on the first surface of thesecond substantially transparent polymer layer, wherein thesubstantially non-transparent polymer layer encloses the center portionsof the first and the second substantially transparent polymer layers,and wherein the substantially non-transparent polymer layer comprises atwo-dimensional shape representing an analog code.

In some embodiments, the magnetic, substantially non-transparent layeris etched by wet etching. In some embodiments, the magnetic,substantially non-transparent layer comprises nickel. In someembodiments, the magnetic, substantially non-transparent layer isbetween about 50 nm and about 10 μm in thickness. In some embodiments,the magnetic, substantially non-transparent layer is less than about 0.1μm in thickness. In some embodiments, the magnetic, substantiallynon-transparent layer comprises an asymmetry for orienting the analogcode of the substantially non-transparent polymer layer. In someembodiments, the two-dimensional shape of the substantiallynon-transparent polymer layer is generated by lithography. In someembodiments, the two-dimensional shape of the substantiallynon-transparent polymer layer comprises a gear shape comprising aplurality of gear teeth, and wherein the analog code is represented byone or more aspects selected from the group consisting of the height ofone or more gear teeth of the plurality, the width of one or more gearteeth of the plurality, the number of gear teeth in the plurality, andthe arrangement of one or more gear teeth within the plurality. In someembodiments, the plurality of gear teeth comprises one or more gearteeth that are between about 1 μm and about 10 μm wide. In someembodiments, the plurality of gear teeth comprises one or more gearteeth that are between about 1 μm and about 10 μm tall. In someembodiments, the plurality of gear teeth comprises two or more gearteeth that are spaced between about 1 μm and about 10 μm apart. In someembodiments, the method further comprises (f) before step (a),depositing a sacrificial layer on a substrate; (g) creating one or morecolumn-shaped holes in the sacrificial layer using lithography; (h)depositing a third substantially transparent polymer layer in the one ormore column-shaped holes in the sacrificial layer, wherein the firstsubstantially transparent polymer layer is deposited in step (a) on topof the third substantially transparent polymer layer and the sacrificiallayer; (i) after step (e), depositing using lithography one or morecolumns comprising the substantially transparent polymer on the firstsurface of the second substantially transparent polymer layer at aportion not covered by the substantially non-transparent polymer layer;(j) dissolving the sacrificial layer in a solvent; and (k) removing thesubstrate. In some embodiments, the method further comprises: (f) beforestep (a), depositing a sacrificial layer on a substrate; (g) as part ofstep (a), depositing the substantially transparent polymer layer on thesacrificial layer; (h) after step (e), dissolving the sacrificial layerin a solvent; and (i) removing the substrate. In some embodiments, theencoded microcarrier is a substantially circular disc. In someembodiments, the center portion of the first substantially transparentpolymer layer comprises between about 5% and about 90% of the surfacearea of the first substantially transparent polymer layer. In someembodiments, the center portion of the first substantially transparentpolymer layer comprises about 25% of the surface area of the firstsubstantially transparent polymer layer. In some embodiments, theencoded microcarrier is less than about 200 μm in diameter. In someembodiments, the encoded microcarrier is about 50 μm in diameter. Insome embodiments, the encoded microcarrier is less than about 50 μm inthickness. In some embodiments, the encoded microcarrier is about 10 μmin thickness. In some embodiments, the method further comprises: (f)coupling a capture agent for capturing an analyte to at least one of thefirst surface of the second substantially transparent polymer layer andthe second surface of the first substantially transparent polymer layerin at least the center portion. In some embodiments, the substantiallytransparent polymer of the first or the second substantially transparentpolymer layer comprises an epoxide, and coupling the capture agentcomprises: (i) reacting the substantially transparent polymer of thefirst and/or the second substantially transparent polymer layers with aphotoacid generator and light to generate a cross-linked polymer,wherein the light is of a wavelength that activates the photoacidgenerator; and (ii) reacting the epoxide of the cross-linked polymerwith a compound comprising an amine and a carboxyl, wherein the amine ofthe compound reacts with the epoxide to form a compound-coupled,cross-linked polymer; and (iii) reacting the carboxyl of thecompound-coupled, cross-linked polymer with the capture agent to couplethe capture agent to at least one of the first surface of the secondsubstantially transparent polymer layer and the second surface of thefirst substantially transparent polymer layer in at least the centerportion. In some embodiments, the carboxyl of the compound-coupled,cross-linked polymer reacts with a primary amine of the capture agent.In some embodiments, the substantially transparent polymer of the firstand/or the second substantially transparent polymer layers compriseSU-8. In some embodiments, the analyte is selected from the groupconsisting of a DNA molecule, a DNA-analog-molecule, an RNA-molecule, anRNA-analog-molecule, a polynucleotide, a protein, an enzyme, a lipid, aphospholipid, a carbohydrate moiety, a polysaccharide, an antigen, avirus, a cell, an antibody, a small molecule, a bacterial cell, acellular organelle, and an antibody fragment. In some embodiments, thecapture agent for capturing the analyte is selected from the groupconsisting of a DNA molecule, a DNA-analog-molecule, an RNA-molecule, anRNA-analog-molecule, a polynucleotide, a protein, an enzyme, a lipid, aphospholipid, a carbohydrate moiety, a polysaccharide, an antigen, avirus, a cell, an antibody, a small molecule, a bacterial cell, acellular organelle, and an antibody fragment.

In another aspect, provided herein is an encoded microcarrier producedby the method of any of the above embodiments.

In another aspect, provided herein is a method of making an encodedmicrocarrier, comprising: (a) depositing a sacrificial layer on asubstrate; (b) depositing on the sacrificial layer a substantiallynon-transparent polymer layer having an outline, a first surface, and asecond surface, the first and the second surfaces being parallel to eachother, wherein the second surface is affixed to the sacrificial layer;(c) shaping by lithography the outline of the substantiallynon-transparent polymer layer, wherein the outline is shaped into atwo-dimensional shape representing an analog code; (d) dissolving thesacrificial polymer layer in a solvent; and (e) removing the substrate.In another aspect, provided herein is a method of making an encodedmicrocarrier, comprising: (a) depositing a sacrificial layer on asubstrate; (b) depositing a magnetic layer comprising a magneticmaterial on the sacrificial layer; (c) depositing on the magnetic layera substantially non-transparent polymer layer having an outline, a firstsurface, and a second surface, the first and the second surfaces beingparallel to each other, wherein the second surface is affixed to themagnetic layer; (d) shaping by lithography the outline of thesubstantially non-transparent polymer layer, wherein the outline isshaped into a two-dimensional shape representing an analog code; (e)dissolving the sacrificial polymer layer in a solvent; and (f) removingthe substrate.

In some embodiments, the methods further include (g) after step (b) andbefore step (c), shaping the magnetic layer by lithography. In someembodiments, the magnetic material comprises nickel. In someembodiments, the microcarrier comprises an orientation indicator fororienting the analog code of the substantially non-transparent polymerlayer. In some embodiments, the orientation indicator comprises anasymmetry of the outline of the substantially non-transparent polymerlayer. In some embodiments, the two-dimensional shape of thesubstantially non-transparent polymer layer comprises a gear shapecomprising a plurality of gear teeth, and wherein the analog code isrepresented by one or more aspects selected from the group consisting ofthe height of one or more gear teeth of the plurality, the width of oneor more gear teeth of the plurality, the number of gear teeth in theplurality, and the arrangement of one or more gear teeth within theplurality. In some embodiments, the plurality of gear teeth comprisesone or more gear teeth that are between about 1 μm and about 10 μm wide.In some embodiments, the plurality of gear teeth comprises one or moregear teeth that are between about 1 μm and about 10 μm tall. In someembodiments, the plurality of gear teeth comprises two or more gearteeth that are spaced between about 1 μm and about 10 μm apart. In someembodiments, the microcarrier is a substantially circular disc. In someembodiments, the microcarrier is less than about 200 μm in diameter. Insome embodiments, the microcarrier is about 60 μm in diameter. In someembodiments, the microcarrier is less than about 30 μm in thickness. Insome embodiments, the microcarrier is about 10 μm in thickness. In someembodiments, the method further includes (h) coupling a capture agentfor capturing an analyte to at least one of the first surface and thesecond surface of the substantially non-transparent polymer layer. Insome embodiments, the substantially non-transparent polymer of thesubstantially non-transparent polymer layer comprises an epoxide, andcoupling the capture agent comprises: (i) reacting the substantiallynon-transparent polymer of the substantially non-transparent polymerlayer with a photoacid generator and light to generate a cross-linkedpolymer, wherein the light is of a wavelength that activates thephotoacid generator; and (ii) reacting the epoxide of the cross-linkedpolymer with a compound comprising an amine and a carboxyl, wherein theamine of the compound reacts with the epoxide to form acompound-coupled, cross-linked polymer; and (iii) reacting the carboxylof the compound-coupled, cross-linked polymer with the capture agent tocouple the capture agent to at least one of the first surface of thesecond substantially transparent polymer layer and the second surface ofthe first substantially transparent polymer layer in at least the centerportion. In some embodiments, the analyte is selected from the groupconsisting of a DNA molecule, a DNA-analog-molecule, an RNA-molecule, anRNA-analog-molecule, a polynucleotide, a protein, an enzyme, a lipid, aphospholipid, a carbohydrate moiety, a polysaccharide, an antigen, avirus, a cell, an antibody, a small molecule, a bacterial cell, acellular organelle, and an antibody fragment. In some embodiments, thecapture agent for capturing the analyte is selected from the groupconsisting of a DNA molecule, a DNA-analog-molecule, an RNA-molecule, anRNA-analog-molecule, a polynucleotide, a protein, an enzyme, a lipid, aphospholipid, a carbohydrate moiety, a polysaccharide, an antigen, avirus, a cell, an antibody, a small molecule, a bacterial cell, acellular organelle, and an antibody fragment.

In another aspect, provided herein is an encoded microcarrier producedby the method of any of the above embodiments.

In another aspect, provided herein is a method for detecting two or moreanalytes in a solution, comprising: (a) contacting a solution comprisinga first analyte and a second analyte with a plurality of microcarriers,wherein the plurality of microcarriers comprises at least: (i) a firstmicrocarrier according to any of the above embodiments that specificallycaptures the first analyte, wherein the first microcarrier is encodedwith a first analog code; and (ii) a second microcarrier according toany of the above embodiments that specifically captures the secondanalyte, wherein the second microcarrier is encoded with a second analogcode, and wherein the second analog code is different from the firstanalog code; (b) decoding the first analog code and the second analogcode using analog shape recognition to identify the first microcarrierand the second microcarrier; and (c) detecting an amount of the firstanalyte bound to the first microcarrier and an amount of the secondanalyte bound to the second microcarrier.

In some embodiments, step (b) occurs before step (c). In someembodiments, step (c) occurs before step (b). In some embodiments, step(b) occurs simultaneously with step (c). In some embodiments, decodingthe first analog code and the second analog code comprises: (i)illuminating the first and second microcarriers by passing light throughthe substantially transparent portions of the first and secondmicrocarriers and/or the surrounding solution, wherein the light failsto pass through the substantially non-transparent portions of the firstand second microcarriers to generate a first analog-coded light patterncorresponding to the first microcarrier and a second analog-coded lightpattern corresponding to the second microcarrier; (ii) imaging the firstanalog-coded light pattern to generate a first analog-coded image andimaging the second analog-coded light pattern to generate a secondanalog-coded image; and (iii) using analog shape recognition to matchthe first analog-coded image with the first analog code and to match thesecond analog-coded image with the second analog code. In someembodiments, detecting the amount of the first analyte bound to thefirst microcarrier and the amount of the second analyte bound to thesecond microcarrier comprises: (i) after step (a), incubating the firstand the second microcarriers with a detection agent, wherein thedetection agent binds the first analyte captured by the firstmicrocarrier and the second analyte captured by the second microcarrier;and (ii) measuring the amount of detection agent bound to the first andthe second microcarriers. In some embodiments, the detection agent is afluorescent detection agent, and the amount of detection agent bound tothe first and the second microcarriers is measured by fluorescencemicroscopy. In some embodiments, the detection agent is a luminescentdetection agent, and the amount of detection agent bound to the firstand the second microcarriers is measured by luminescence microscopy. Insome embodiments, the solution comprises a biological sample. In someembodiments, the biological sample is selected from the group consistingof blood, urine, sputum, bile, cerebrospinal fluid, interstitial fluidof skin or adipose tissue, saliva, tears, bronchial-alveolar lavage,oropharyngeal secretions, intestinal fluids, cervico-vaginal or uterinesecretions, and seminal fluid.

In another aspect, provided herein is a kit or article of manufacturecomprising a plurality of microcarriers, wherein the plurality ofmicrocarriers comprises at least: (a) a first microcarrier according toany of the above embodiments that specifically captures a first analyte,wherein the first microcarrier is encoded with a first analog code; and(b) a second microcarrier according to any of the above embodiments thatspecifically captures a second analyte, wherein the second microcarrieris encoded with a second analog code, and wherein the second analog codeis different from the first analog code.

In some embodiments, the kit or article of manufacture further comprisesa detection agent for detecting an amount of the first analyte bound tothe first microcarrier and an amount of the second analyte bound to thesecond microcarrier. In some embodiments, the kit or article ofmanufacture further comprises instructions for using the kit to detectthe first analyte and the second analyte.

It is to be understood that one, some, or all of the properties of thevarious embodiments described herein may be combined to form otherembodiments of the present invention. These and other aspects of theinvention will become apparent to one of skill in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A & 1B show two views of an exemplary microcarrier.

FIGS. 1C & 1D show an exemplary assay for analyte detection using anexemplary microcarrier.

FIGS. 2A & 2B show two views of an exemplary microcarrier.

FIG. 3 shows an exemplary analog encoding scheme that includes multipleshape variation points for generating unique analog codes.

FIG. 4A shows three examples of microcarriers, each having a uniqueanalog code.

FIG. 4B shows examples of microcarriers with a unique analog code, inaccordance with some embodiments.

FIGS. 5A & 5B show two views of an exemplary microcarrier.

FIGS. 6A & 6B show two views of an exemplary microcarrier.

FIG. 6C shows the dimensions of an exemplary analog code. Dimensions arebased on μm units.

FIG. 7 shows an exemplary microcarrier.

FIG. 8A shows an exemplary microcarrier that includes an asymmetricstart position as an orientation indicator.

FIG. 8B shows an exemplary analog encoding scheme that includes multipleshape variation points for generating unique analog codes.

FIGS. 9A-9C show two views of an exemplary microcarrier (FIG. 9A andFIG. 9B), along with a depiction of an optional feature (FIG. 9C).

FIG. 10 shows a method for producing an exemplary microcarrier.

FIGS. 11A & 11B show a method for producing an exemplary microcarrier.

FIGS. 12A-12E show a method for producing an exemplary microcarrier.

FIGS. 13A-13C show a method for producing an exemplary microcarrier.

DETAILED DESCRIPTION

In one aspect, provided herein are encoded microcarriers for analytedetection in multiplex assays. In some embodiments, the microcarriersinclude (a) a substantially transparent polymer layer having a firstsurface and a second surface, the first and the second surfaces beingparallel to each other; (b) a substantially non-transparent polymerlayer, where the substantially non-transparent polymer layer is affixedto the first surface of the substantially transparent polymer layer andencloses a center portion of the substantially transparent polymerlayer, and where the substantially non-transparent polymer layercomprises a two-dimensional shape representing an analog code; and (c) acapture agent for capturing an analyte, where the capture agent iscoupled to at least one of the first surface and the second surface ofthe substantially transparent polymer layer in at least the centerportion of the substantially transparent polymer layer. In otherembodiments, the microcarriers include (a) a substantiallynon-transparent polymer layer having a first surface and a secondsurface, the first and the second surfaces being parallel to each other,wherein an outline of the substantially non-transparent polymer layercomprises a two-dimensional shape that represents an analog code; and(b) a capture agent for capturing an analyte, wherein the capture agentis coupled to at least one of the first surface and the second surfaceof the substantially non-transparent polymer layer in at least a centerportion of the substantially non-transparent polymer layer.

In another aspect, provided herein are methods of making encodedmicrocarriers. In some embodiments, the methods include (a) depositing asubstantially transparent polymer layer, wherein the substantiallytransparent polymer layer has a first surface and a second surface, thefirst and the second surfaces being parallel to each other; (b)depositing a magnetic, substantially non-transparent layer on the firstsurface of the substantially transparent polymer layer; (c) etching themagnetic, substantially non-transparent layer to remove a portion of themagnetic, substantially non-transparent layer that is deposited over acenter portion of the substantially transparent polymer layer; (d)depositing a second substantially transparent polymer layer over themagnetic, substantially non-transparent layer, where the secondsubstantially transparent polymer layer has a first surface and a secondsurface, the first and the second surfaces being parallel to each other,where the second surface is affixed to the magnetic, substantiallynon-transparent layer, and where the second substantially transparentpolymer layer is aligned with the first substantially transparentpolymer layer and has a center portion that is aligned with the centerportion of the substantially transparent polymer layer; and (e)depositing a substantially non-transparent polymer layer on the firstsurface of the second substantially transparent polymer layer, where thesubstantially non-transparent polymer layer encloses the center portionsof the first and the second substantially transparent polymer layers,and where the substantially non-transparent polymer layer comprises atwo-dimensional shape representing an analog code. In other embodiments,the methods include (a) depositing a sacrificial layer on a substrate;(b) depositing on the sacrificial layer a substantially non-transparentpolymer layer having an outline, a first surface, and a second surface,the first and the second surfaces being parallel to each other, wherethe second surface is affixed to the sacrificial layer; (c) shaping bylithography the outline of the substantially non-transparent polymerlayer, where the outline is shaped into a two-dimensional shaperepresenting an analog code; (d) dissolving the sacrificial polymerlayer in a solvent; and (e) removing the substrate. In otherembodiments, the methods include (a) depositing a sacrificial layer on asubstrate; (b) depositing a magnetic layer comprising a magneticmaterial on the sacrificial layer; (c) depositing on the magnetic layera substantially non-transparent polymer layer having an outline, a firstsurface, and a second surface, the first and the second surfaces beingparallel to each other, wherein the second surface is affixed to themagnetic layer; (d) shaping by lithography the outline of thesubstantially non-transparent polymer layer, where the outline is shapedinto a two-dimensional shape representing an analog code; (d) dissolvingthe sacrificial polymer layer in a solvent; and (e) removing thesubstrate. Further provided herein are encoded microcarriers produced bythe methods disclosed herein.

In still another aspect, provided herein are methods for detecting twoor more analytes in a solution by (a) contacting a solution comprising afirst analyte and a second analyte with a plurality of microcarriers,where the plurality of microcarriers includes at least: (i) a firstmicrocarrier of the present disclosure that specifically captures thefirst analyte, where the first microcarrier is encoded with a firstanalog code; and (ii) a second microcarrier of the present disclosurethat specifically captures the second analyte, where the secondmicrocarrier is encoded with a second analog code, and where the secondanalog code is different from the first analog code; (b) decoding thefirst analog code and the second analog code using analog shaperecognition to identify the first microcarrier and the secondmicrocarrier; and (c) detecting an amount of the first analyte bound tothe first microcarrier and an amount of the second analyte bound to thesecond microcarrier.

In yet another aspect, provided herein are kits or articles ofmanufacture for conducting a multiplex assay including a plurality ofmicrocarriers. The plurality of microcarriers includes at least (a) afirst microcarrier of the present disclosure that specifically capturesa first analyte, where the first microcarrier is encoded with a firstanalog code; and (b) a second microcarrier of the present disclosurethat specifically captures a second analyte, where the secondmicrocarrier is encoded with a second analog code, and where the secondanalog code is different from the first analog code.

I. General Techniques

The practice of the techniques described herein will employ, unlessotherwise indicated, conventional techniques in polymer technology,microfabrication, micro-electro-mechanical systems (MEMS) fabrication,photolithography, microfluidics, organic chemistry, biochemistry,oligonucleotide synthesis and modification, bioconjugate chemistry,nucleic acid hybridization, molecular biology, microbiology, genetics,recombinant DNA, and related fields as are within the skill of the art.The techniques are described in the references cited herein and arefully explained in the literature.

For molecular biology and recombinant DNA techniques, see, for example,(Maniatis, T. et al. (1982), Molecular Cloning: A Laboratory Manual,Cold Spring Harbor; Ausubel, F. M. (1987), Current Protocols inMolecular Biology, Greene Pub. Associates and Wiley-Interscience;Ausubel, F. M. (1989), Short Protocols in Molecular Biology: ACompendium of Methods from Current Protocols in Molecular Biology,Greene Pub. Associates and Wiley-Interscience; Sambrook, J. et al.(1989), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor;Innis, M. A. (1990), PCR Protocols: A Guide to Methods and Applications,Academic Press; Ausubel, F. M. (1992), Short Protocols in MolecularBiology: A Compendium of Methods from Current Protocols in MolecularBiology, Greene Pub. Associates; Ausubel, F. M. (1995), Short Protocolsin Molecular Biology: A Compendium of Methods from Current Protocols inMolecular Biology, Greene Pub. Associates; Innis, M. A. et al. (1995),PCR Strategies, Academic Press; Ausubel, F. M. (1999), Short Protocolsin Molecular Biology: A Compendium of Methods from Current Protocols inMolecular Biology, Wiley, and annual updates.

For DNA synthesis techniques and nucleic acids chemistry, see forexample, Gait, M. J. (1990), Oligonucleotide Synthesis: A PracticalApproach, IRL Press; Eckstein, F. (1991), Oligonucleotides andAnalogues: A Practical Approach, IRL Press; Adams, R. L. et al. (1992),The Biochemistry of the Nucleic Acids, Chapman & Hall; Shabarova, Z. etal. (1994), Advanced Organic Chemistry of Nucleic Acids, Weinheim;Blackburn, G. M. et al. (1996), Nucleic Acids in Chemistry and Biology,Oxford University Press; Hermanson, G. T. (1996), BioconjugateTechniques, Academic Press).

For microfabrication, see for example, (Campbell, S. A. (1996), TheScience and Engineering of Microelectronic Fabrication, OxfordUniversity Press; Zaut, P. V. (1996), Microarray Fabrication: aPractical Guide to Semiconductor Processing, Semiconductor Services;Madou, M. J. (1997), Fundamentals of Microfabrication, CRC Press;Rai-Choudhury, P. (1997). Handbook of Microlithography, Micromachining,& Microfabrication: Microlithography).

II. Definitions

Before describing the invention in detail, it is to be understood thatthis invention is not limited to particular compositions or biologicalsystems, which can, of course, vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting.

The term “microcarrier” as used herein may refer to a physical substrateonto which a capture agent may be coupled. A microcarrier of the presentdisclosure may take any suitable geometric form or shape. In someembodiments, the microcarrier may be disc-shaped. Typically the form orshape of a microcarrier will include at least one dimension on the orderof 10⁻⁴ to 10⁻⁷ m (hence the prefix “micro”).

The term “polymer” as used herein may refer to any macromolecularstructure comprising repeated monomers. A polymer may be natural (e.g.,found in nature) or synthetic (e.g., man-made, such as a polymercomposed of non-natural monomer(s) and/or polymerized in a configurationor combination not found in nature).

The terms “substantially transparent” and “substantiallynon-transparent” as used herein may refer to the ability of light (e.g.,of a particular wavelength, such as infrared, visible, UV, and so forth)to pass through a substrate, such as a polymer layer. A substantiallytransparent polymer may refer to one that is transparent, translucent,and/or pervious to light, whereas a substantially non-transparentpolymer may refer to one that reflects and/or absorbs light. It is to beappreciated that whether a material is substantially transparent orsubstantially non-transparent may depend upon the wavelength and/orintensity of light illuminating the material, as well as the meansdetecting the light traveling through the material (or a decrease orabsence thereof). In some embodiments, a substantially non-transparentmaterial causes a perceptible decrease in transmitted light as comparedto the surrounding material or image field, e.g., as imaged by lightmicroscopy (e.g., bright field, dark field, phase contrast, differentialinterference contrast (DIC), Nomarski interference contrast (NIC),Nomarski, Hoffman modulation contrast (HMC), or fluorescencemicroscopy). In some embodiments, a substantially transparent materialallows a perceptible amount of transmitted light to pass through thematerial, e.g., as imaged by light microscopy (e.g., bright field, darkfield, phase contrast, differential interference contrast (DIC),Nomarski interference contrast (NIC), Nomarski, Hoffman modulationcontrast (HMC), or fluorescence microscopy).

The term “analog code” as used herein may refer to any code in which theencoded information is represented in a non-quantized and/ornon-discrete manner, e.g., as opposed to a digital code. For example, adigital code is sampled at discrete positions for a limited set ofvalues (e.g., 0/1 type values), whereas an analog code may be sampled ata greater range of positions (or as a continuous whole) and/or maycontain a wider set of values (e.g., shapes). In some embodiments, ananalog code may be read or decoded using one or more analog shaperecognition techniques.

The term “capture agent” as used herein is a broad term and is used inits ordinary sense to refer to any compound or substance capable ofspecifically recognizing an analyte of interest. In some embodiments,specific recognition may refer to specific binding. Non-limitingexamples of capture agents include, for example, a DNA molecule, aDNA-analog-molecule, an RNA-molecule, an RNA-analog-molecule, apolynucleotide, a protein, an enzyme, a lipid, a phospholipid, acarbohydrate moiety, a polysaccharide, an antigen, a virus, a cell, anantibody, a small molecule, a bacterial cell, a cellular organelle, andan antibody fragment.

“Analyte,” as used herein, is a broad term and is used in its ordinarysense as a substance the presence, absence, or quantity of which is tobe determined, including, without limitation, to refer to a substance orchemical constituent in a sample such as a biological sample or cell orpopulation of cells that can be analyzed. An analyte can be a substancefor which a naturally occurring binding member exists, or for which abinding member can be prepared. Non-limiting examples of analytesinclude, for example, antibodies, antibody fragments, antigens,polynucleotides (such as a DNA molecule, DNA-analog-molecule,RNA-molecule, or RNA-analog-molecule), polypeptides, proteins, enzymes,lipids, phospholipids, carbohydrate moieties, polysaccharides, smallmolecules, organelles, hormones, cytokines, growth factors, steroids,vitamins, toxins, drugs, and metabolites of the above substances, aswell as cells, bacteria, viruses, fungi, algae, fungal spores and thelike.

The term “antibody” is used in the broadest sense and includesmonoclonal antibodies (including full length antibodies which have animmunoglobulin Fc region), polyclonal antibodies, multispecificantibodies (e.g., bispecific antibodies, diabodies, and single-chainmolecules), as well as antibody fragments (e.g., Fab, F(ab′)₂, and Fv).

As used herein, “sample” refers to a composition containing a material,such as a molecule, to be detected. In one embodiment, the sample is a“biological sample” (i.e., any material obtained from a living source(e.g. human, animal, plant, bacteria, fungi, protist, virus)). Thebiological sample can be in any form, including solid materials (e.g.tissue, cell pellets and biopsies) and biological fluids (e.g. urine,blood, saliva, lymph, tears, sweat, prostatic fluid, seminal fluid,semen, bile, mucus, amniotic fluid and mouth wash (containing buccalcells)). Solid materials typically are mixed with a fluid. Sample canalso refer to an environmental sample such as water, air, soil, or anyother environmental source.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a molecule”optionally includes a combination of two or more such molecules, and thelike.

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse.

It is understood that aspects and embodiments of the invention describedherein include “comprising,” “consisting,” and “consisting essentiallyof” aspects and embodiments.

III. Encoded Microcarriers

Provided herein are encoded microcarriers suitable for analytedetection, e.g., multiplex analyte detection. Multiple configurationsfor encoded microcarriers are contemplated, described, and exemplifiedherein.

In some aspects, provided herein are encoded microcarriers thatcomprise: a substantially transparent polymer layer having a firstsurface and a second surface, the first and the second surfaces beingparallel to each other; a substantially non-transparent polymer layer,wherein the substantially non-transparent polymer layer is affixed tothe first surface of the substantially transparent polymer layer andencloses a center portion of the substantially transparent polymerlayer, and wherein the substantially non-transparent polymer layercomprises a two-dimensional shape representing an analog code; and acapture agent for capturing an analyte, wherein the capture agent iscoupled to at least one of the first surface and the second surface ofthe substantially transparent polymer layer in at least the centerportion of the substantially transparent polymer layer. Thus, themicrocarrier contains at least two layers: one of which is substantiallytransparent, and the other of which is a substantially non-transparent,two-dimensional shape that represents an analog code. Advantageously,these microcarriers may employ a variety of two-dimensional shapes whilestill retaining a uniform overall form (e.g., the perimeter of thesubstantially transparent polymer layer) for uniformity of aspectsincluding, for example, overall dimensions, physical properties, and/orbehavior in solution. Examples of this type of microcarrier and aspectsthereof are illustrated in FIGS. 1A-5B.

In some embodiments, the microcarrier further includes a magnetic,substantially non-transparent layer affixed to a surface of thesubstantially transparent polymer layer that encloses the center portionof the substantially transparent polymer layer. In some embodiments, themagnetic, substantially non-transparent layer is between thesubstantially non-transparent polymer layer and the center portion ofthe substantially transparent polymer layer.

In some embodiments, the microcarrier further includes a secondsubstantially transparent polymer layer aligned with and affixed to thefirst substantially transparent polymer layer. In some embodiments, thefirst and second substantially transparent polymer layers each have acenter portion, and the center portions of both the first and secondsubstantially transparent polymer layers are aligned. In someembodiments, the microcarrier further includes a magnetic, substantiallynon-transparent layer that encloses the center portions of both thefirst and second substantially transparent polymer layers. In someembodiments, the magnetic, substantially non-transparent layer isaffixed between the first and second substantially transparent polymerlayers. In some embodiments, the magnetic, substantially non-transparentlayer is between the substantially non-transparent polymer layer and thecenter portions of both the first and second substantially transparentpolymer layers.

In some embodiments, the magnetic, substantially non-transparent layeris between about 50 nm and about 10 μm in thickness. In someembodiments, the thickness of the magnetic, substantiallynon-transparent layer is less than about any of the followingthicknesses (in nm): 10000, 9500, 9000, 8500, 8000, 7500, 7000, 6500,6000, 5500, 5000, 4500, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 950,900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250,200, 150, or 100. In some embodiments, the thickness of the magnetic,substantially non-transparent layer is greater than about any of thefollowing thicknesses (in nm): 50, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000,2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000,8500, 9000, or 9500. That is, the thickness of the magnetic,substantially non-transparent layer may be any of a range of thicknesses(in nm) having an upper limit of 10000, 9500, 9000, 8500, 8000, 7500,7000, 6500, 6000, 5500, 5000, 4500, 4000, 3500, 3000, 2500, 2000, 1500,1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350,300, 250, 200, 150, or 100 and an independently selected lower limit of50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500,5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, or 9500, whereinthe lower limit is less than the upper limit.

In some embodiments, the magnetic, substantially non-transparent layeris about 0.1 μm in thickness. In some embodiments, the magnetic,substantially non-transparent layer is about 50 nm, about 100 nm, about150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about400 nm, about 450 nm, about 500 nm, about 550 nm, about 600 nm, about650 nm, about 700 nm, about 750 nm, about 800 nm, about 850 nm, about900 nm, about 950 nm, about 1 μm, about 1.5 μm, about 2 μm, about 2.5μm, about 3 μm, about 3.5 μm, about 4 μm, about 4.5 μm, about 5 μm,about 5.5 μm, about 6 μm, about 6.5 μm, about 7 μm, about 7.5 μm, about8 μm, about 8.5 μm, about 9 μm, about 9.5 μm, or about 10 μm inthickness. In some embodiments, the thickness of the magnetic,substantially non-transparent layer is about 0.01 μm, about 0.02 μm,about 0.03 μm, about 0.04 μm, about 0.05 μm, about 0.06 μm, about 0.07μm, about 0.08 μm, about 0.09 μm, about 0.1 μm, about 0.11 μm, about0.12 μm, about 0.13 μm, about 0.14 μm, about 0.15 μm, about 0.16 μm,about 0.17 μm, about 0.18 μm, about 0.19 μm, about 0.20 μm, about 0.25μm, about 0.30 μm, about 0.35 μm, about 0.40 μm, about 0.45 μm, or about0.50 μm.

In some embodiments, the microcarrier further includes an orientationindicator for orienting the analog code of the substantiallynon-transparent polymer layer. Any feature of the microcarrier that isvisible and/or detectable by imaging (e.g., a form of microscopic orother imaging described herein) and/or by image recognition software mayserve as an orientation indicator. An orientation indicator may serve asa point of reference, e.g., for an image recognition algorithm, toorient the image of an analog code in a uniform orientation (i.e., theshape of the substantially non-transparent polymer layer).Advantageously, this simplifies image recognition, as the algorithmwould only need to compare the image of a particular analog code againsta library of analog codes in the same orientation, and not against alibrary including all analog codes in all possible orientations. In someembodiments, the orientation indicator may be independent of thesubstantially non-transparent polymer layer. For example, it may beformed as a part of a magnetic layer and/or substantially transparentpolymer layer. In other embodiments, the orientation indicator may beformed as part of the substantially non-transparent polymer layer. Insome embodiments, the orientation indicator comprises an asymmetry ofthe magnetic, substantially non-transparent layer (e.g., as illustratedby gap 210 in FIG. 2A).

In some embodiments, the microcarrier further includes one or morecolumns projecting from a surface of the microcarrier (e.g., the topand/or bottom surface of the microcarrier). As used herein, a “column”may refer to any geometric shape that projects from the microcarriersurface and does not necessarily denote any regularity in dimensions,nor any cylindrical character. For example, the outer surface of acolumn may or may not be parallel with the microcarrier surface.Examples of columnar shapes that may project from a microcarrier includewithout limitation a rectangular prism, a triangle, a pyramid, a cube, acylinder, a sphere or half-sphere, a cone, and so forth. In someembodiments, the one or more columns are not within a center portion ofthe first and/or the second substantially transparent polymer layer. Insome embodiments, the one or more columns may project from anoutside-facing surface (e.g., a surface not affixed to another layer) ofone or more of the first and the second substantially transparentpolymer layers. It is to be noted that any descriptions of microcarrierthickness herein do not include the one or more columns in the stateddimensions. That is to say, microcarrier thickness as described hereinis independent of any optional columns projecting therefrom.

In some embodiments, the one or more columns are between about 1 μm andabout 10 μm tall. In some embodiments, the one or more columns are about1 μm tall, about 1.5 μm tall, about 2 μm tall, about 2.5 μm tall, about3 μm tall, about 3.5 μm tall, about 4 μm tall, about 4.5 μm tall, about5 μm tall, about 5.5 μm tall, about 6 μm tall, about 6.5 μm tall, about7 μm tall, about 7.5 μm tall, about 8 μm tall, about 8.5 μm tall, about9 μm tall, about 9.5 μm tall, or about 10 μm tall. In some embodiments,the one or more columns are less than about any of the following heights(in μm): 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3,2.5, 2, or 1.5. In some embodiments, the one or more columns are greaterthan about any of the following heights (in μm): 1, 1.5, 2, 2.5, 3, 3.5,4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5. That is, the one ormore columns can be any of a range of heights having an upper limit of10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or1.5 and an independently selected lower limit of 1, 1.5, 2, 2.5, 3, 3.5,4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5, wherein the lowerlimit is less than the upper limit.

In some embodiments, the one or more columns may be cylindrical inshape. In some embodiments, the one or more columns have a diameterbetween about 1 μm and about 10 μm. In some embodiments, the one or morecolumns have a diameter of about 1 μm, about 1.5 μm, about 2 μm, about2.5 μm, about 3 μm, about 3.5 μm, about 4 μm, about 4.5 μm, about 5 μm,about 5.5 μm, about 6 μm, about 6.5 μm, about 7 μm, about 7.5 μm, about8 μm, about 8.5 μm, about 9 μm, about 9.5 μm, or about 10 μm. In someembodiments, the one or more columns have a diameter less than about anyof the following lengths (in μm): 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6,5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5. In some embodiments, the one ormore columns have a diameter greater than about any of the followinglengths (in μm): 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5,8, 8.5, 9, or 9.5. That is, the one or more columns can have any of arange of diameters having an upper limit of 10, 9.5, 9, 8.5, 8, 7.5, 7,6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5 and an independentlyselected lower limit of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5,7, 7.5, 8, 8.5, 9, or 9.5, wherein the lower limit is less than theupper limit. In other embodiments, the one or more columns may haveroughly the same width as any diameter described supra, or a range ofwidths roughly the same as any range of diameters described supra, butthe one or more columns may adopt the shape of an elliptical cylinder,parabolic cylinder, hyperbolic cylinder, or any other cylindrical orpolyhedral shape described herein or known in the art.

In other aspects, provided herein are encoded microcarriers thatcomprise: a substantially non-transparent polymer layer having a firstsurface and a second surface, the first and the second surfaces beingparallel to each other, wherein an outline of the substantiallynon-transparent polymer layer comprises a two-dimensional shape thatrepresents an analog code; and a capture agent for capturing an analyte,wherein the capture agent is coupled to at least one of the firstsurface and the second surface of the substantially non-transparentpolymer layer in at least a center portion of the substantiallynon-transparent polymer layer. Thus, the microcarrier is encoded by theshape (e.g., outline) of the microcarrier itself: a two-dimensionalshape that represents an analog code. Advantageously, thesemicrocarriers may be manufactured efficiently and with high precision,allowing for highly accurate decoding and cost-efficient production.Examples of this type of microcarrier and aspects thereof areillustrated in FIGS. 6A-9C.

In some embodiments, the microcarrier further includes one or morecolumns projecting from a surface of the substantially non-transparentpolymer layer. As described in greater detail supra, a “column” mayrefer to any geometric shape that projects from the microcarrier surfaceand does not necessarily denote any regularity in columnar dimension(s).Any of the exemplary columnar shapes described above may be used.

In some embodiments, the one or more columns are between about 1 μm andabout 10 μm tall. In some embodiments, the one or more columns are about1 μm tall, about 1.5 μm tall, about 2 μm tall, about 2.5 μm tall, about3 μm tall, about 3.5 μm tall, about 4 μm tall, about 4.5 μm tall, about5 μm tall, about 5.5 μm tall, about 6 μm tall, about 6.5 μm tall, about7 μm tall, about 7.5 μm tall, about 8 μm tall, about 8.5 μm tall, about9 μm tall, about 9.5 μm tall, or about 10 μm tall. In some embodiments,the one or more columns are less than about any of the following heights(in μm): 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3,2.5, 2, or 1.5. In some embodiments, the one or more columns are greaterthan about any of the following heights (in μm): 1, 1.5, 2, 2.5, 3, 3.5,4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5. That is, the one ormore columns can be any of a range of heights having an upper limit of10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or1.5 and an independently selected lower limit of 1, 1.5, 2, 2.5, 3, 3.5,4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5, wherein the lowerlimit is less than the upper limit.

In some embodiments, the one or more columns may be cylindrical inshape. In some embodiments, the one or more columns have a diameterbetween about 1 μm and about 10 μm. In some embodiments, the one or morecolumns have a diameter of about 1 μm, about 1.5 μm, about 2 μm, about2.5 μm, about 3 μm, about 3.5 μm, about 4 μm, about 4.5 μm, about 5 μm,about 5.5 μm, about 6 μm, about 6.5 μm, about 7 μm, about 7.5 μm, about8 μm, about 8.5 μm, about 9 μm, about 9.5 μm, or about 10 μm. In someembodiments, the one or more columns have a diameter less than about anyof the following lengths (in μm): 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6,5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5. In some embodiments, the one ormore columns have a diameter greater than about any of the followinglengths (in μm): 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5,8, 8.5, 9, or 9.5. That is, the one or more columns can have any of arange of diameters having an upper limit of 10, 9.5, 9, 8.5, 8, 7.5, 7,6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5 and an independentlyselected lower limit of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5,7, 7.5, 8, 8.5, 9, or 9.5, wherein the lower limit is less than theupper limit. In other embodiments, the one or more columns may haveroughly the same width as any diameter described supra, or a range ofwidths roughly the same as any range of diameters described supra, butthe one or more columns may adopt the shape of an elliptical cylinder,parabolic cylinder, hyperbolic cylinder, or any other cylindrical orpolyhedral shape described herein or known in the art.

In some embodiments, the microcarrier further includes a magnetic layercomprising a magnetic material affixed to a surface of the substantiallynon-transparent polymer layer. In some embodiments, the magnetic layerdoes not extend beyond the two-dimensional shape of the substantiallynon-transparent polymer layer. That is to say, if the outline of thesubstantially non-transparent polymer layer were to be imaged, theresulting image would not be altered by the presence or absence of themagnetic layer. In some embodiments, the magnetic layer may include theone or more columns described above. That is, the one or more columnsdescribed above may be made of a magnetic material described herein.

In some embodiments, the microcarrier further includes an orientationindicator for orienting the analog code of the substantiallynon-transparent polymer layer. Any feature of the microcarrier that isvisible and/or detectable by imaging (e.g., a form of microscopic orother imaging described herein) and/or by image recognition software mayserve as an orientation indicator. An orientation indicator may serve asa point of reference, e.g., for an image recognition algorithm, toorient the image of an analog code in a uniform orientation (i.e., theshape of the substantially non-transparent polymer layer).Advantageously, this simplifies image recognition, as the algorithmwould only need to compare the image of a particular analog code againsta library of analog codes in the same orientation, and not against alibrary including all analog codes in all possible orientations. In someembodiments, the orientation indicator comprises an asymmetry of theoutline of the substantially non-transparent polymer layer. For example,the orientation indicator may comprise a visible feature, such as anasymmetry, of the outline of the microcarrier (e.g., as illustrated bystart positions 804 and 904 in FIGS. 8A and 9A).

Any of the microcarriers described herein may include one or more of thefeatures, elements, or aspects described below. In addition, one or moreof the features, elements, or aspects described below may adoptdifferent characteristics depending on the embodiment of themicrocarrier, e.g., as described above.

In some embodiments, a substantially transparent polymer of the presentdisclosure comprises an epoxy-based polymer. Suitable epoxy-basedpolymers for fabrication of the compositions described herein include,but are not limited to, the EPON™ family of epoxy resins provided byHexion Specialty Chemicals, Inc. (Columbus, Ohio) and any number ofepoxy resins provided by The Dow Chemical Company (Midland, Mich.). Manyexamples of suitable polymers are commonly known in the art, includingwithout limitation SU-8, EPON 1002F, EPON 165/154, and a poly(methylmethacrylate)/poly(acrylic acid) block copolymer (PMMA-co-PAA). Foradditional polymers, see, for example, Warad, IC Packaging: PackageConstruction Analysis in Ultra Small IC Packaging, LAP LAMBERT AcademicPublishing (2010); The Electronic Packaging Handbook, CRC Press(Blackwell, ed.), (2000); and Pecht et al., Electronic PackagingMaterials and Their Properties, CCR Press, 1^(st) ed., (1998). Thesetypes of materials have the advantage of not swelling in aqueousenvironments which ensures that uniform microcarrier size and shape aremaintained within the population of microcarriers. In some embodiments,the substantially transparent polymer is a photoresist polymer. In someembodiments, the epoxy-based polymer is an epoxy-based, negative-tone,near-UV photoresist. In some embodiments, the epoxy-based polymer isSU-8.

In some embodiments, the substantially non-transparent polymer is apolymer described herein (e.g., SU-8) mixed with one or morenon-transparent or colored dye(s). In other embodiments, thesubstantially non-transparent polymer is a black matrix resist. Anyblack matrix resist known in the art may be used; see, e.g., U.S. Pat.No. 8,610,848 for exemplary black matrix resists and methods relatedthereto. In some embodiments, the black matrix resist may be aphotoresist colored with a black pigment, e.g., as patterned on thecolor filter of an LCD as part of a black matrix. Black matrix resistsmay include without limitation those sold by Toppan Printing Co.(Tokyo), Tokyo OHKA Kogyo (Kawasaki), and Daxin Materials Corp.(Taichung City, Taiwan).

In some embodiments, reference may be made to a center portion of one ormore polymer layers. A center portion of the present disclosure may takeany shape. In some embodiments, the shape of the center portion mayreflect or correspond to the shape (e.g., outline) of the correspondingpolymer layer. In other embodiments, the shape of the center portion maybe independent of the shape (e.g., outline) of the corresponding polymerlayer. For example, a center portion of a circular microcarrier surfacemay be circular in some embodiments and square in other embodiments. Acenter portion of a square microcarrier surface may be square in someembodiments and circular in other embodiments.

In some embodiments, a center portion of a polymer layer of the presentdisclosure is about 5%, about 7%, about 10%, about 15%, about 20%, about25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%,about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, orabout 90% of the surface area of the polymer layer. In some embodiments,a center portion of a polymer layer of the present disclosure is lessthan about any of the following fractions of the substantiallytransparent polymer layer (in %): 90, 85, 80, 75, 70, 65, 60, 55, 50,45, 40, 35, 30, 25, 20, 15, 10, or 7. In some embodiments, a centerportion of a polymer layer of the present disclosure is greater thanabout any of the following fractions of the substantially transparentpolymer layer (in %): 5, 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, or 85. That is, the fraction of the polymer layersurface area included in the center portion may be any of a range ofpercentages having an upper limit of 90, 85, 80, 75, 70, 65, 60, 55, 50,45, 40, 35, 30, 25, 20, 15, 10, or 7 and an independently selected lowerlimit of 5, 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, or 85, wherein the lower limit is less than the upper limit. In someembodiments, the center portion of a polymer layer comprises about 25%of the surface area of the polymer layer. In some embodiments, a centerportion of a microcarrier surface includes the entire surface minus anoutline portion of the microcarrier.

As described above, a microcarrier of the present disclosure may furtherinclude a magnetic layer, which may adopt a variety of shapes asdescribed herein. In some embodiments, the magnetic layer may be asubstantially non-transparent layer. In some embodiments, the magneticlayer may comprise a magnetic material. A magnetic layer of the presentdisclosure may be made of any suitable magnetic material, such as amaterial with paramagnetic, ferromagnetic, or ferrimagnetic properties.Examples of magnetic materials include without limitation iron, nickel,cobalt, and some rare earth metals (e.g., gadolinium, dysprosium,neodymium, and so forth), as well as alloys thereof. In someembodiments, the magnetic material comprises nickel, including withoutlimitation elemental nickel and magnetic nickel alloys such as alnicoand permalloy. The inclusion of a magnetic layer in a microcarrier ofthe present disclosure may be advantageous, e.g., in facilitatingmagnetic separation, which may be useful for washing, collecting, andotherwise manipulating one or more microcarriers.

As described above, in some embodiments, the magnetic layer may beaffixed to a surface of the substantially transparent polymer layer andenclose a center portion of the substantially transparent polymer layer.In other embodiments, as described above, the magnetic layer may includeone or more columns; i.e., the one or more columns described above maybe made of a magnetic material described herein.

In some embodiments, a microcarrier of the present disclosure may beencoded with a substantially non-transparent layer that constitutes atwo-dimensional shape. For example, as described above, thetwo-dimensional shape may constitute the shape of a substantiallynon-transparent layer that contrasts with a substantially transparentlayer of the microcarrier, or it may constitute the shape of themicrocarrier itself (e.g., the perimeter). Any two-dimensional shapethat can encompass a plurality of resolvable and distinctive varietiesmay be used. In some embodiments, the two-dimensional shape comprisesone or more of linear, circular, elliptical, rectangular, quadrilateral,or higher polygonal aspects, elements, and/or shapes.

In some embodiments, the two-dimensional shape of the substantiallynon-transparent polymer layer comprises a gear shape. A gear shape asused herein may refer to a plurality of shapes (e.g., gear teeth)arrayed on the perimeter of a substantially round, elliptical, orcircular body, where at least two of the shapes of the plurality arespatially separated. In some embodiments, the gear shape comprises aplurality of gear teeth. In some embodiments, the analog code isrepresented by one or more aspects selected from the height of one ormore gear teeth of the plurality, the width of one or more gear teeth ofthe plurality, the number of gear teeth in the plurality, and thearrangement of one or more gear teeth within the plurality.Advantageously, a gear shape encompasses multiple aspects, including theheight of gear teeth, the width of gear teeth, the number of gear teeth,and the arrangement of gear teeth, that may be varied in order togenerate a large diversity of potential unique two-dimensional shapes.It is to be appreciated, however, that since the gear shapes of thepresent disclosure are used for encoding and are not required tophysically intermesh with another gear (e.g., as with mechanical gearsthat transmit torque), gear teeth of the present disclosure are notconstrained by the need for identical or intermeshing shapes, eitherwithin one gear shape or between multiple gear shapes. As such, thevariety of shapes that may be considered a gear tooth of the presentdisclosure is significantly greater than with a mechanical gear.

In some embodiments, the plurality of gear teeth comprises one or moregear teeth that are between about 1 μm and about 10 μm wide. In someembodiments, the plurality of gear teeth comprises one or more gearteeth that are about 1 μm wide, about 1.5 μm wide, about 2 μm wide,about 2.5 μm wide, about 3 μm wide, about 3.5 μm wide, about 4 μm wide,about 4.5 μm wide, about 5 μm wide, about 5.5 μm wide, about 6 μm wide,about 6.5 μm wide, about 7 μm wide, about 7.5 μm wide, about 8 μm wide,about 8.5 μm wide, about 9 μm wide, about 9.5 μm wide, or about 10 μmwide. In some embodiments, the plurality of gear teeth comprises one ormore gear teeth that are less than about any of the following widths (inμm): 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2,or 1.5. In some embodiments, the plurality of gear teeth comprises oneor more gear teeth that are greater than about any of the followingwidths (in μm): 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5,8, 8.5, 9, or 9.5. That is, the plurality of gear teeth may comprise oneor more gear teeth that can be any of a range of widths having an upperlimit of 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3,2.5, 2, or 1.5 and an independently selected lower limit of 1, 1.5, 2,2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5, whereinthe lower limit is less than the upper limit.

In some embodiments, the plurality of gear teeth comprises one or moregear teeth that are between about 1 μm and about 10 μm tall. In someembodiments, the plurality of gear teeth comprises one or more gearteeth that are about 1 μm tall, about 1.5 μm tall, about 2 μm tall,about 2.5 μm tall, about 3 μm tall, about 3.5 μm tall, about 4 μm tall,about 4.5 μm tall, about 5 μm tall, about 5.5 μm tall, about 6 μm tall,about 6.5 μm tall, about 7 μm tall, about 7.5 μm tall, about 8 μm tall,about 8.5 μm tall, about 9 μm tall, about 9.5 μm tall, or about 10 μmtall. In some embodiments, the plurality of gear teeth comprises one ormore gear teeth that are less than about any of the following heights(in μm): 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3,2.5, 2, or 1.5. In some embodiments, the plurality of gear teethcomprises one or more gear teeth that are greater than about any of thefollowing heights (in μm): 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, or 9.5. That is, the plurality of gear teeth maycomprise one or more gear teeth that can be any of a range of heightshaving an upper limit of 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5,4.5, 4, 3.5, 3, 2.5, 2, or 1.5 and an independently selected lower limitof 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or9.5, wherein the lower limit is less than the upper limit. It is to beappreciated that a gear tooth may have different measurable heights,depending on the point of reference, if the adjacent perimeter segmentsfrom which the gear tooth extends are uneven (see, e.g., gear tooth 602in FIG. 6C, which may be 4 or 6.5 μm tall, depending on the point ofreference).

In some embodiments, the plurality of gear teeth comprises one or moregear teeth that are spaced between about 1 μm and about 10 μm apart. Insome embodiments, the plurality of gear teeth comprises one or more gearteeth that are spaced about 1 μm apart, about 1.5 μm apart, about 2 μmapart, about 2.5 μm apart, about 3 μm apart, about 3.5 μm apart, about 4μm apart, about 4.5 μm apart, about 5 μm apart, about 5.5 μm apart,about 6 μm apart, about 6.5 μm apart, about 7 μm apart, about 7.5 μmapart, about 8 μm apart, about 8.5 μm apart, about 9 μm apart, about 9.5μm apart, or about 10 μm apart. In some embodiments, the plurality ofgear teeth comprises one or more gear teeth that are spaced less thanabout any of the following widths apart (in μm): 10, 9.5, 9, 8.5, 8,7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5. In someembodiments, the plurality of gear teeth comprises one or more gearteeth that are spaced greater than about any of the following widthsapart (in μm): 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5,8, 8.5, 9, or 9.5. That is, the plurality of gear teeth may comprise oneor more gear teeth that can be spaced any of a range of widths aparthaving an upper limit of 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5,4.5, 4, 3.5, 3, 2.5, 2, or 1.5 and an independently selected lower limitof 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or9.5, wherein the lower limit is less than the upper limit.

In some embodiments, a microcarrier of the present disclosure is asubstantially circular disc. As used herein, a substantially circularshape may refer to any shape having a roughly identical distance betweenall of the points of the shape's perimeter and the shape's geometriccenter. In some embodiments, a shape is considered to be substantiallycircular if the variation among any of the potential radii connectingthe geometric center and a given point on the perimeter exhibit 10% orlesser variation in length. As used herein, a substantially circulardisc may refer to any substantially circular shape wherein the thicknessof the shape is significantly less than its diameter. For example, insome embodiments, the thickness of a substantially circular disc may beless than about 50%, less than about 40%, less than about 30%, less thanabout 20%, less than about 15%, less than about 10%, or less than about5% of its diameter. In certain embodiments, the thickness of thesubstantially circular disc may about 20% of its diameter. It is to beappreciated that the microcarriers of the present disclosure whoseoutline is a gear shape may also be considered substantially circulardiscs; for example, the shape of the microcarrier excluding the one ormore gear teeth may comprise a substantially circular disc.

In some embodiments, the microcarrier is less than about 200 μm indiameter. For example, in some embodiments, the diameter of themicrocarrier is less than about 200 μm, less than about 180 μm, lessthan about 160 μm, less than about 140 μm, less than about 120 μm, lessthan about 100 μm, less than about 80 μm, less than about 60 μm, lessthan about 40 μm, or less than about 20 μm.

In some embodiments, the diameter of the microcarrier is about 180 μm,about 160 μm, about 140 μm, about 120 μm, about 100 μm, about 90 μm,about 80 μm, about 70 μm, about 60 μm, about 50 μm, about 40 μm, about30 μm, about 20 μm, or about 10 μm. In certain embodiments, themicrocarrier is about 60 μm in diameter.

In some embodiments, the microcarrier is less than about 50 μm inthickness. For example, in some embodiments, the thickness of themicrocarrier is less than about 70 μm, about 60 μm, about 50 μm, about40 μm, about 30 μm, less than about 25 μm, less than about 20 μm, lessthan about 15 μm, less than about 10 μm, or less than about 5 μm. Insome embodiments, the thickness of the microcarrier is less than aboutany of the following thicknesses (in μm): 70, 65, 60, 55, 50, 45, 40,35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2. In some embodiments,the thickness of the microcarrier is greater than about any of thefollowing thicknesses (in μm): 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, or 65. That is, the thickness of themicrocarrier may be any of a range of thicknesses (in μm) having anupper limit of 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8,7, 6, 5, 4, 3, or 2 and an independently selected lower limit of 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 65,wherein the lower limit is less than the upper limit.

In some embodiments, the thickness of the microcarrier is about 50 μm,about 45 μm, about 40 μm, about 35 μm, about 30 μm, about 25 μm, about20 μm, about 19 μm, about 18 μm, about 17 μm, about 16 μm, about 15 μm,about 14 μm, about 13 μm, about 12 μm, about 11 μm, about 10 μm, about 9μm, about 8 μm, about 7 μm, about 6 μm, about 5 μm, about 4 μm, about 3μm, about 2 μm, or about 1 μm. In certain embodiments, the microcarrieris about 10 μm in thickness.

In some aspects, a microcarrier of the present disclosure can comprise acapture agent. In some embodiments, the capture agent for a particularmicrocarrier species may be a “unique capture agent,” e.g., a captureagent is associated with a particular microcarrier species having aparticular identifier (e.g., analog code). The capture agent can be anybiomolecule or a chemical compound capable of binding one or moreanalytes (such as a biomolecule or chemical compound) present in thesolution. Examples of biomolecule capture agents include, but are notlimited to, a DNA molecule, a DNA-analog-molecule, an RNA-molecule, anRNA-analog-molecule, a polynucleotide, a protein, an enzyme, a lipid, aphospholipid, a carbohydrate moiety, a polysaccharide, an antigen, avirus, a cell, an antibody, a small molecule, a bacterial cell, acellular organelle, and an antibody fragment. Examples of chemicalcompound capture agents include, but are not limited to, individualcomponents of chemical libraries, small molecules, or environmentaltoxins (for example, pesticides or heavy metals).

In some embodiments, the capture agent is coupled to a surface of themicrocarrier (in some embodiments, in at least a center portion of themicrocarrier surface). In some embodiments, the capture agent can bechemically attached to the microcarrier. In other embodiments, thecapture agent can be physically absorbed to the surface of themicrocarrier. In some embodiments, the attachment linkage between thecapture agent and the microcarrier surface can be a covalent bond. Inother embodiments, the attachment linkage between the capture agent andthe microcarrier surface can be a non-covalent bond including, but notlimited to, a salt bridge or other ionic bond, one or more hydrogenbonds, hydrophobic interactions, Van der Waals force, London dispersionforce, a mechanical bond, one or more halogen bonds, aurophilicity,intercalation, or stacking.

In some aspects, more than one (such as two, three, four, five, six,seven, eight, nine, or ten) capture agents for the same analyte can eachbe associated with a microcarrier described herein. In this embodiment,each capture agent for a particular analyte binds to the analyte with adifferent affinity as measured by the dissociation constant ofanalyte/capture agent binding. Accordingly, within a plurality ofmicrocarriers in a composition, there can be two or more subpopulationsof microcarriers with capture agents that bind to the same analyte, butwherein the capture agents associated with each subpopulation bind tothe analyte with a different affinity. In some embodiments, thedissociation constant of the analyte for any of the capture agents isnot greater than 10⁻⁶ M, such as 10⁻⁷ M or 10⁻⁸M. In other embodiments,the dissociation constant of the analyte for any of the capture agentsis from about 10⁻¹⁰ M to about 10⁻⁶ M, such from about 10⁻¹⁰ M to about10⁻⁷ M, about 10⁻¹⁰ M to about 10⁻⁸ M, about 10⁻¹⁰ M to about 10⁻⁹ M,about 10⁻⁹ M to about 10⁻⁶ M, about 10⁻⁹M to about 10⁻⁷ M, about 10⁻⁹ Mto about 10⁻⁸ M, about 10⁻⁸ M to about 10⁻⁶ M, or about 10⁻⁸ M to about10⁻⁷ M. In some embodiments, the dissociation constant of the analytefor any two capture agents differs by as much as about 3 log₁₀, such asby as much as about 2.5 log₁₀, 2 log₁₀, 1.5 log₁₀, or 1 log₁₀.

In some embodiments, an analyte of the present disclosure is coupled toa microcarrier for the capture of one or more analytes. In someembodiments, the one or more analytes may be captured from a sample,such as a biological sample described herein. In some embodiments, ananalyte may include without limitation a DNA molecule, aDNA-analog-molecule, an RNA-molecule, an RNA-analog-molecule, apolynucleotide, a protein, an enzyme, a lipid, a phospholipid, acarbohydrate moiety, a polysaccharide, an antigen, a virus, a cell, anantibody, a small molecule, a bacterial cell, a cellular organelle, andan antibody fragment. In other embodiments, the analyte is a chemicalcompound (such as a small molecule chemical compound) capable of bindingto the capture agent such as individual components of chemicallibraries, small molecules, or environmental toxins (for example,pesticides or heavy metals).

In some aspects, the analytes in a sample (such as a biological sample)can be labeled with a signal-emitting entity capable of emitting adetectable signal upon binding to the capture agent. In someembodiments, the signal-emitting entity can be colorimetric based. Inother embodiments, the signal-emitting entity can be fluorescence-basedincluding, but not limited to, phycoerythrin, blue fluorescent protein,green fluorescent protein, yellow fluorescent protein, cyan fluorescentprotein, and derivatives thereof. In other embodiments, thesignal-emitting entity can be radioisotope based, including, but notlimited to, molecules labeled with ³²P, ³³P, ²²Na, ³⁶Cl, ²H, ³H, ³⁵S,and ¹²³I. In other embodiments, the signal-emitting entity islight-based including, but not limited to, luciferase (e.g.,chemiluminescence-based), horseradish peroxidase, alkaline phosphatase,and derivatives thereof. In some embodiments, the biomolecules orchemical compounds present in the sample can be labeled with thesignal-emitting entity prior to contact with the microcarrier. In otherembodiments, the biomolecules or chemical compounds present in thesample can be labeled with the signal-emitting entity subsequent tocontact with the microcarrier.

IV. Methods of Making Encoded Microcarriers

Certain aspects of the present disclosure relate to methods for makingan encoded microcarrier, e.g., a microcarrier described herein. Themethods for making an encoded microcarrier may include one or more ofthe microcarrier features or aspects described herein, e.g., in sectionIII above and/or the Examples that follow.

In some embodiments, the methods include depositing a substantiallytransparent polymer layer, where the substantially transparent polymerlayer has a first surface and a second surface, the first and the secondsurfaces being parallel to each other In some embodiments, the first andthe second surfaces that are parallel to each other may be the top andbottom surface of a single layer. Any suitable substantially transparentpolymer known in the art or described herein may be used. In someembodiments, the substantially transparent polymer layer is depositedusing spin coating.

In some embodiments, the substantially transparent polymer layer may bedeposited on a substrate. Suitable substrates may include substratesused in standard semiconductor and/or micro-electro-mechanical systems(MEMS) fabrication techniques. In some embodiments, the substrate maycomprise glass, silicon, quartz, plastic, polyethylene terephthalate(PET), an indium tin oxide (ITO) coating, or the like.

In some embodiments, a sacrificial layer may be deposited on thesubstrate, e.g., a substrate as described above. In some embodiments,the sacrificial layer may be made of a polymer, including withoutlimitation polyvinyl alcohol (PVA) or OmniCoat™ (MicroChem; Newton,Mass.). Sacrificial layers may be applied, used, and dissolved orstripped, e.g., according to manufacturer's instructions.

In some embodiments, a substantially transparent polymer layer of thepresent disclosure is deposited on a sacrificial layer. To generate aplanar microcarrier surface using a substantially transparent polymerlayer, the substantially transparent polymer layer may be deposited ontoa planar sacrificial layer. To generate a microcarrier surface with oneor more columns projecting therefrom, a sacrificial layer (e.g., onedeposited onto a substrate) may be patterned with one or morecolumn-shaped holes or void areas, for example by using a standardlithographic process. In some embodiments, a substantially transparentpolymer layer may be deposited over the sacrificial layer and optionalsubstrate such that the layer is deposited in the one or morecolumn-shaped holes or void areas. In some embodiments, anothersubstantially transparent polymer layer may then be deposited over thesacrificial layer and the one or more column-shaped holes or void areasfilled with the first substantially transparent polymer layer.

In some embodiments, a magnetic, substantially non-transparent layer ofthe present disclosure is deposited on the first surface of thesubstantially transparent polymer layer. In some embodiments, themagnetic, substantially non-transparent layer is deposited bysputtering. The magnetic, substantially non-transparent layer may bemade of, e.g., any of the magnetic materials described herein. Forexample, in some embodiments, the magnetic, substantiallynon-transparent layer comprises nickel (e.g., elemental nickel, or analloy thereof).

In some embodiments, the magnetic, substantially non-transparent layermay be etched to remove a portion of the magnetic, substantiallynon-transparent layer that is deposited over a center portion of thesubstantially transparent polymer layer. The magnetic, substantiallynon-transparent layer may be etched by any means known in the art. Forexample, in some embodiments, the magnetic, substantiallynon-transparent layer is etched by conventional wet etching. Exemplarydimensions, shapes, and optional asymmetries for a magnetic,substantially non-transparent layer are provided supra.

In some embodiments, a second substantially transparent polymer layer ofthe present disclosure is deposited over the magnetic, substantiallynon-transparent layer. In some embodiments, the second substantiallytransparent polymer layer has a first surface and a second surface thatare parallel to each other (e.g., the top and bottom surface of a singlelayer). In some embodiments, the second surface is affixed to themagnetic, substantially non-transparent layer. In some embodiments, thesecond substantially transparent polymer layer is aligned with the firstsubstantially transparent polymer layer and has a center portion that isaligned with the center portion of the substantially transparent polymerlayer. Exemplary dimensions for the center portion of a substantiallytransparent polymer layer are provided supra.

In some embodiments, a substantially non-transparent polymer layer ofthe present disclosure is deposited on the first surface of the secondsubstantially transparent polymer layer. In some embodiments, thesubstantially non-transparent polymer layer encloses the center portionsof the first and the second substantially transparent polymer layers. Insome embodiments, the substantially non-transparent polymer layercomprises a two-dimensional shape representing an analog code. Any ofthe two-dimensional shapes described or exemplified herein may be used,e.g., a gear shape of the present disclosure. In some embodiments, thesubstantially non-transparent polymer layer is deposited over the secondsubstantially transparent polymer layer and etched (e.g., using astandard lithographic process) into the desired two-dimensional shape.

In some embodiments, one or more columns may be deposited on thesubstantially transparent polymer, e.g., on the first surface of thesecond substantially transparent polymer layer at a portion not coveredby the substantially non-transparent polymer layer. The one or morecolumns may be deposited as described herein, e.g., using a standardlithographic process.

In some embodiments that employ an optional sacrificial layer and/orsubstrate of the present disclosure, the sacrificial layer may bedissolved or stripped, and/or the substrate may be removed, using asolvent. A variety of solvents useful for fabrication (e.g., in standardsemiconductor or MEMS fabrication processes, such as photoresistremoval) are known in the art. In some embodiments, the solvent is aphotoresist stripper solvent, such as a DMSO- or 1-methyl-2-pyrrolidon(NMP)-based solvent. In some embodiments, the solvent is an AZ®photoresist stripper, such as AZ® 300T (AZ Electronic Materials;Somerville, N.J.).

In some embodiments, the methods include depositing a sacrificial layerof the present disclosure on a substrate of the present disclosure.Sacrificial layers, substrates, and suitable deposition methods aredescribed, e.g., as above.

In some embodiments, a substantially non-transparent polymer layer ofthe present disclosure is deposited on the sacrificial layer. In someembodiments, the substantially non-transparent polymer layer has a firstand a second surface that are parallel to each other (e.g., the top andbottom surface of a single layer). In some embodiments, the secondsurface is affixed to the sacrificial layer.

In some embodiments, the outline of the substantially non-transparentpolymer layer is shaped into a two-dimensional shape representing ananalog code, e.g., as described herein. The substantiallynon-transparent polymer layer may be shaped by any method known in theart or described herein, e.g., using a standard lithographic processincluding but not limited to spin coating, soft baking, UV exposure,etching, and hard baking.

In some embodiments, the sacrificial layer may be dissolved or stripped,and/or the substrate may be removed, using a solvent, e.g., as describedabove.

In other embodiments, a magnetic layer comprising a magnetic material ofthe present disclosure is deposited on the sacrificial layer. Exemplarymagnetic materials, magnetic layer shapes/dimensions, and depositionmethods related thereto are provided supra. For example, in someembodiments, the magnetic layer may be shaped into one or more columns,e.g., as illustrated by column 906. In other embodiments, the magneticlayer may be between two non-transparent polymer layers, e.g., embeddedas illustrated by magnetic layer 704. The magnetic material may contain,e.g., any of the magnetic materials described herein. For example, insome embodiments, the magnetic material comprises nickel (e.g.,elemental nickel, or an alloy thereof).

In some embodiments, a substantially non-transparent polymer layer ofthe present disclosure is deposited on the magnetic layer. In someembodiments, the substantially non-transparent polymer layer has a firstand a second surface that are parallel to each other (e.g., the top andbottom surface of a single layer). In some embodiments, a surface (e.g.,the second surface) of the substantially non-transparent polymer layeris affixed to the magnetic layer.

In some embodiments, the outline of the substantially non-transparentpolymer layer is shaped into a two-dimensional shape representing ananalog code, e.g., as described above.

In some embodiments, the sacrificial layer may be dissolved or stripped,and/or the substrate may be removed, using a solvent, e.g., as describedabove.

Exemplary microcarrier shapes, dimensions, and optional featuressuitable for the methods described above are provided throughout thepresent disclosure.

In some embodiments, a capture agent may be coupled to a microcarrier ofthe present disclosure, e.g., a microcarrier described herein and/or amicrocarrier produced by any of the methods described herein. Any of thecapture agents described herein, or any capture agent known in the artsuitable for capturing an analyte described herein, may find use in themethods and/or microcarriers of the present disclosure.

In some embodiments, the capture agent may be coupled to a polymer layerof the present disclosure, e.g., a substantially transparent orsubstantially non-transparent polymer layer described herein. In someembodiments, the capture agent may be coupled to one or both of a firstor a second surface of the polymer layer. In some embodiments, thecapture agent may be coupled to at least the center portion of thepolymer layer (e.g., a center portion as described herein). In someembodiments, the polymer comprises an epoxy-based polymer or otherwisecontains an epoxide group.

In some embodiments, coupling the capture agent involves reacting thepolymer with a photoacid generator and light to generate a cross-linkedpolymer. In some embodiments, the light is of a wavelength thatactivates the photoacid generator, e.g., UV or near-UV light. Photoacidgenerators are commercially available from Sigma-Aldrich (St. Louis) andBASF (Ludwigshafen). Any suitable photoacid generator known in the artmay be used, including without limitation triphenyl or triaryl sulfoniumhexafluoroantimonate; triarylsulfonium hexafluorophosphate;triphenylsulfonium perfluoro-1-butanesulfonate; triphenylsulfoniumtriflate; Tris(4-tert-butylphenyl)sulfonium perfluoro-1-butanesulfonateor triflate; Bis(4-tert-butylphenyl)iodonium-containing photoacidgenerators such as Bis(4-tert-butylphenyl)iodoniumperfluoro-1-butanesulfonate, p-toluenesulfonate, and triflate;Boc-methoxyphenyldiphenylsulfonium triflate;(tert-Butoxycarbonylmethoxynaphthyl)-diphenylsulfonium triflate;(4-tert-Butylphenyl)diphenylsulfonium triflate; diphenyliodoniumhexafluorophosphate, nitrate, perfluoro-1-butanesulfonate, triflate, orp-toluenesulfonate; (4-fluorophenyl)diphenylsulfonium triflate;N-hydroxynaphthalimide triflate;N-hydroxy-5-norbornene-2,3-dicarboximide perfluoro-1-butanesulfonate;(4-iodophenyl)diphenylsulfonium triflate;(4-methoxyphenyl)diphenylsulfonium triflate;2-(4-methoxystyryl)-4,6-bis(trichloromethyl)-1,3,5-triazine;(4-methylphenyl)diphenylsulfonium triflate; (4-methylthiophenyl)methylphenyl sulfonium triflate; (4-phenoxyphenyl)diphenylsulfonium triflate;(4-phenylthiophenyl)diphenylsulfonium triflate; or any of the photoacidgenerators described inproduct-finder.basficom/group/corporate/product-finder/de/literature-documentiBrand+Irgacure-Brochure—Photoacid+Generator+Selection+Guide-English.pdf.In some embodiments, the photoacid generator is a sulfonium-containingphotoacid generator.

In some embodiments, coupling the capture agent involves reacting anepoxide of the cross-linked polymer with a functional group such as anamine, carboxyl, thiol, or the like. Alternatively, the epoxy group onthe surface can be oxidized to hydroxyl group, which is subsequentlyused as initiation sites for graft polymerization of water solublepolymers such as poly(acrylic acid). The carboxyl groups in poly(acrylicacid) are then used to form covalent bonds with amino or hydroxyl groupsin capture agents.

In some embodiments, coupling the capture agent involves reacting anepoxide of the cross-linked polymer with a compound that contains anamine and a carboxyl. In some embodiments, the amine of the compoundreacts with the epoxide to form a compound-coupled, cross-linkedpolymer. Without wishing to be bound to theory, it is thought that thecapture agent may be coupled to the polymer before the polymer iscross-linked; however, this may reduce the uniformity of the resultingsurface. Any compound with a primary amine and a carboxyl group may beused. Compounds may include without limitation glycine, amino undecanoicacid, amino caproic acid, acrylic acid, 2-carboxyethyl acrylic acid,4-vinylbenzoic acid, 3-acrylamido-3-methyl-1-butanoic acid, glycidylmethacrylate, and the like. In some embodiments, the carboxyl of thecompound-coupled, cross-linked polymer reacts with an amine (e.g., aprimary amine) of the capture agent to couple the capture agent to thesubstantially transparent polymer.

Descriptions of various capture agents and analytes suitable for themethods described above may be found throughout the present disclosure,e.g., in section III above and/or the Examples that follow.

V. Multiplex Assays

Certain aspects of the present disclosure relate to methods fordetecting analytes in a solution by using an encoded microcarrier, e.g.,a microcarrier described herein. The methods for analyte detection usingan encoded microcarrier that includes one or more of the microcarrierfeatures or aspects described herein, e.g., in sections III and IV aboveand/or the Examples that follow. Advantageously, these encodedmicrocarriers allow for analyte detection in improved multiplex assayswith a large number of potential unique microcarriers and reducedrecognition error, as compared to traditional multiplex assays. Theanalyte detection methods used herein may be performed in any suitableassay vessel known in the art, for example a microplate, petri dish, orany number of other well-known assay vessels.

In some embodiments, the methods for detecting analytes in a solutioncomprise contacting a solution comprising a first analyte and a secondanalyte with a plurality of microcarriers, where the plurality ofmicrocarriers comprises at least a first microcarrier of the presentdisclosure that specifically captures the first analyte and is encodedwith a first analog code, and a second microcarrier of the presentdisclosure that specifically captures the second analyte and is encodedwith a second analog code; decoding the first analog code and the secondanalog code using analog shape recognition to identify the firstmicrocarrier and the second microcarrier; and detecting an amount of thefirst analyte bound to the first microcarrier and an amount of thesecond analyte bound to the second microcarrier.

In some embodiments, the methods include contacting a solutioncomprising a first analyte and a second analyte with a plurality ofmicrocarriers. In some embodiments, the plurality of microcarriers mayinclude a first microcarrier of the present disclosure that specificallycaptures the first analyte (e.g., using a capture agent, coupled to themicrocarrier, specific for the first analyte), where the firstmicrocarrier is encoded with a first analog code; and a secondmicrocarrier of the present disclosure that specifically captures thesecond analyte (e.g., using a capture agent, coupled to themicrocarrier, specific for the second analyte), where the secondmicrocarrier is encoded with a second analog code different from thefirst analog code. In some embodiments, the first and second analytesmay be different. In other embodiments, the first and second analytesmay be the same, e.g., the first and second microcarriers mayredundantly recognize the same analyte (this may be useful, e.g., forquality control purposes), or they may recognize distinct regions of thesame analyte (e.g., antibodies recognizing different epitopes of thesame antigen).

The methods of the present disclosure may be used to detect analytes inany suitable solution. In some embodiments, the solution comprises abiological sample. Examples of biological samples include withoutlimitation blood, urine, sputum, bile, cerebrospinal fluid, interstitialfluid of skin or adipose tissue, saliva, tears, bronchial-alveolarlavage, oropharyngeal secretions, intestinal fluids, cervico-vaginal oruterine secretions, and seminal fluid. In some embodiments, thebiological sample may be from a human. In other embodiments, thesolution comprises a sample that is not a biological sample, such as anenvironmental sample, a sample prepared in a laboratory (e.g., a samplecontaining one or more analytes that have been prepared, isolated,purified, and/or synthesized), a fixed sample (e.g., a formalin-fixed,paraffin-embedded or FFPE sample), and so forth.

In some embodiments, the analysis is multiplexed, that is, each solution(e.g., a sample) is analyzed so that a signal from the signal emittingentity is detected by the reaction detection system for at least 2analytes of interest, at least 3 analytes of interest, at least 4analytes of interest, at least 5 analytes of interest, at least 10analytes of interest, at least 15 analytes of interest, at least 20analytes of interest, at least 25 analytes of interest, at least 30analytes of interest, at least 35 analytes of interest, at least 40analytes of interest, at least 45 analytes of interest, or at least 50analytes of interest, or more.

In some embodiments, the methods include decoding the first analog codeand the second analog code using analog shape recognition to identifythe first microcarrier and the second microcarrier. Conceptually, thisdecoding may involve imaging the analog code of each microcarrier (e.g.,in a solution or sample), comparing each image against a library ofanalog codes, and matching each image to an image from the library, thuspositively identifying the code. Optionally, as described herein, whenusing microcarriers that include an orientation indicator (e.g., anasymmetry), the decoding may further include a step of rotating eachimage to align with a particular orientation (based in part, e.g., onthe orientation indicator). For example, if the orientation indicatorincludes a gap, the image could be rotated until the gap reaches apredetermined position or orientation (e.g., a 0° position of theimage).

Various shape recognition software, tools, and methods are known in theart. Examples of such APIs and tools include without limitationMicrosoft® Research FaceSDK, OpenBR, Face and Scene Recognition fromReKognition, Betaface API, and various ImageJ plugins. In someembodiments, the analog shape recognition may include without limitationimage processing steps such as foreground extraction, shape detection,thresholding (e.g., automated or manual image thresholding), and thelike.

It will be appreciated by one of skill in the art that the methods andmicrocarriers described herein may be adapted for various imagingdevices, including without limitation a microscope, plate reader, andthe like. In some embodiments, decoding the analog codes may includeilluminating the first and second microcarriers by passing light throughthe substantially transparent portions (e.g., substantially transparentpolymer layer(s)) of the first and second microcarriers and/or thesurrounding solution. The light may then fail to pass through, or passthrough with a lower intensity or other appreciable difference, thesubstantially non-transparent portions (e.g., substantiallynon-transparent polymer layer(s)) of the first and second microcarriersto generate a first analog-coded light pattern corresponding to thefirst microcarrier and a second analog-coded light pattern correspondingto the second microcarrier.

As described supra, any type of light microscopy may be used for themethods of the present disclosure, including without limitation one ormore of: bright field, dark field, phase contrast, differentialinterference contrast (DIC), Nomarski interference contrast (NIC),Nomarski, Hoffman modulation contrast (HMC), or fluorescence microscopy.In certain embodiments, the analog codes may be decoded using brightfield microscopy, and analyte(s) may be detected using fluorescencemicroscopy.

In some embodiments, decoding the analog codes may further includeimaging the first analog-coded light pattern to generate a firstanalog-coded image and imaging the second analog-coded light pattern togenerate a second analog-coded image. That is to say, the pattern ofimaged light may correspond to the pattern of substantiallytransparent/substantially non-transparent areas of the microcarrier,thus producing an image of the analog codes. This imaging may includesteps including without limitation capturing the image, thresholding theimage, and any other image processing step desired to achieve moreaccurate, precise, or robust imaging of the analog codes.

In some embodiments, decoding the analog codes may further include usinganalog shape recognition to match the first analog-coded image with thefirst analog code and to match the second analog-coded image with thesecond analog code. In some embodiments, an image may be matched with ananalog code (e.g., an image file from a library of image files, witheach image file corresponding to a unique two-dimensional shape/analogcode) within a predetermined threshold, e.g., that tolerates apredetermined amount of deviation or mismatch between the image and theexemplar analog code image. Such a threshold may be empiricallydetermined and may naturally be based on the particular type oftwo-dimensional shapes used for the analog codes and the extent ofvariation among the set of potential two-dimensional shapes.

In some embodiments, the methods include detecting an amount of thefirst analyte bound to the first microcarrier and an amount of thesecond analyte bound to the second microcarrier. Any suitable analytedetection technique(s) known in the art may be used. For example, insome embodiments, the first and the second microcarriers may beincubated with one or more detection agents. In some embodiments, theone or more detection agents bind the first analyte captured by thefirst microcarrier and the second analyte captured by the secondmicrocarrier. In some embodiments, the methods further include measuringthe amount of detection agent bound to the first and the secondmicrocarriers.

In some embodiments, the analytes in a solution (such as a biologicalsample) can be labeled with a detection agent (e.g., a signal-emittingentity) capable of emitting a detectable signal upon binding to thecapture agent. In some embodiments, the detection agent can becolorimetric based. In other embodiments, the detection agent can befluorescence-based including, but not limited to, phycoerythrin, bluefluorescent protein, green fluorescent protein, yellow fluorescentprotein, cyan fluorescent protein, and derivatives thereof. In otherembodiments, the detection agent can be radioisotope based, including,but not limited to, molecules labeled with ³²P, ³³P, ²²Na, ³⁶Cl, ²H, ³H,³⁵S, and ¹²³I. In other embodiments, the detection agent is light-basedincluding, but not limited to, luciferase (e.g.chemiluminescence-based), horseradish peroxidase, alkaline phosphataseand derivatives thereof. In some embodiments, the biomolecules orchemical compounds present in the solution can be labeled with thedetection agent prior to contact with the microcarrier composition. Inother embodiments, the biomolecules or chemical compounds present in thesolution can be labeled with the detection agent subsequent to contactwith the microcarrier composition. In yet other embodiments, thedetection agent may be coupled to a molecule or macromolecular structurethat specifically binds the analyte of interest, e.g., a DNA molecule, aDNA-analog-molecule, an RNA-molecule, an RNA-analog-molecule, apolynucleotide, a protein, an enzyme, a lipid, a phospholipid, acarbohydrate moiety, a polysaccharide, an antigen, a virus, a cell, anantibody, a small molecule, a bacterial cell, a cellular organelle,and/or an antibody fragment.

In some embodiments, the detection agent is a fluorescent detectionagent, and the amount of detection agent bound to the first and thesecond microcarriers is measured by fluorescence microscopy (e.g., afluorescent microscope or plate reader). In other embodiments, thedetection agent is a luminescent detection agent, and the amount ofdetection agent bound to the first and the second microcarriers ismeasured by luminescence microscopy (e.g., a luminescent microscope orplate reader).

In some embodiments, each analyte/capture agent may be used with aspecific detection agent. As non-limiting examples, the detection agentmay be a detection agent (e.g., a fluorescent, luminescent, enzymatic,or other detection agent) coupled to an antibody that specifically bindsthe analyte; or a ligand or receptor of a ligand-receptor pair, if theanalyte is a cognate ligand/receptor of the ligand-receptor pair. Thistechnique is conceptually similar to a sandwich ELISA or proteinmicroarray that includes a capture and a detection antibody (though itshould be noted in the present case that the agents in this example arenot strictly limited to antibodies). As another non-limiting example,the detection agent may be a fluorescent or other detectable probecoupled to a protein of interest, such as a labeled analyte of interest.For example, a reaction may be used to couple detection agent(s) to oneor more proteins in a solution of interest (e.g., a sample), which wouldthen be captured by the capture agents (conceptually similar to anantigen capture-type of protein microarray).

In other embodiments, multiple unique analytes/capture agents may beused with a universal detection agent. As non-limiting examples, thedetection agent may be an agent that binds to the Fc region of anantibody, if the analyte is an antibody; a fluorescent or otherdetectable probe coupled to an oligonucleotide (e.g., a single strandedoligonucleotide that hybridizes with an analyte), if the analyte is apolynucleotide such as DNA or RNA. The later scenario is conceptuallysimilar to a microarray technique.

In some embodiments, the detecting steps may include one or more washingsteps, e.g., to reduce contaminants, remove any substancesnon-specifically bound to the capture agent and/or microcarrier surface,and so forth. In some embodiments, a magnetic separation step may beused to wash a microcarrier containing a magnetic layer or material ofthe present disclosure. In other embodiments, other separation stepsknown in the art may be used.

In some embodiments, the decoding step(s) may occur after the detectingstep(s). In other embodiments, the decoding step(s) may occur before thedetecting step(s). In still other embodiments, the decoding step(s) mayoccur simultaneously with the detecting step(s).

VI. Kits or Articles of Manufacture

Further provided herein are kits or articles of manufacture containing aplurality of microcarriers of the present disclosure. These kits orarticles of manufacture may find use, inter alia, in conducting amultiplex assay, such as the exemplary multiplex assays described herein(see, e.g., section V above).

In some embodiments, the kits or articles of manufacture may include afirst microcarrier of the present disclosure that specifically capturesa first analyte (e.g., using a capture agent, coupled to themicrocarrier, specific for the first analyte), where the firstmicrocarrier is encoded with a first analog code; and a secondmicrocarrier of the present disclosure that specifically captures thesecond analyte (e.g., using a capture agent, coupled to themicrocarrier, specific for the second analyte), where the secondmicrocarrier is encoded with a second analog code different from thefirst analog code. In some embodiments, the first and second analytesmay be different. In other embodiments, the first and second analytesmay be the same, e.g., the first and second microcarriers mayredundantly recognize the same analyte (this may be useful, e.g., forquality control purposes), or they may recognize distinct regions of thesame analyte (e.g., antibodies recognizing different epitopes of thesame antigen). The kits or articles of manufacture may include any ofthe microcarriers described herein (see, e.g., section III above and theExamples infra) or produced using the methods described herein (see,e.g., section IV above and the Examples infra).

In some embodiments, the kits or articles of manufacture may furtherinclude one or more detection agents of the present disclosure fordetecting an amount of the first analyte bound to the first microcarrierand an amount of the second analyte bound to the second microcarrier. Insome embodiments, the detection agent for the first analyte may be thesame as the detection agent for the second analyte. In otherembodiments, the detection agent for the first analyte may be differentfrom the detection agent for the second analyte.

In some embodiments, the kits or articles of manufacture may furtherinclude instructions for using the kit or articles of manufacture todetect one or more analytes, e.g., the first and the second analyte.These instructions may be for using the kit or article of manufacture,e.g., in any of the methods described herein.

In some embodiments, the kits or articles of manufacture may furtherinclude one or more detection agents (e.g., as described above), alongwith any instructions or reagents suitable for coupling a detectionagent to one or more analytes, or for coupling a detection agent to oneor more macromolecules that recognize an analyte. The kits or articlesof manufacture may further include any additional components for usingthe microcarriers in an assay (e.g., a multiplex assay), includingwithout limitation a plate (e.g., a 96-well or other similarmicroplate), dish, microscope slide, or other suitable assay container;a non-transitory computer-readable storage medium (e.g., containingsoftware and/or other instructions for analog shape or coderecognition); washing agents; buffers; plate sealers; mixing containers;diluents or storage solutions; and the like.

EXAMPLES

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. It is understood that the examples andembodiments described herein are for illustrative purposes only and thatvarious modifications or changes in light thereof will be suggested topersons skilled in the art and are to be included within the spirit andpurview of this application and scope of the appended claims.

Attention is now directed towards microcarriers for multiplex assays(e.g., analyte detection) and their methods of production. The followingExamples illustrate exemplary embodiments of analog-encodedmicrocarriers for analyte detection that may find use, inter alia, inthe methods, assays, and kits or articles of manufacture describedherein. It is to be noted that these exemplary embodiments are in no wayintended to be limiting but are provided to illustrate some of theaspects and features set forth herein.

Example 1 Encoded Microcarriers with a Two-Dimensional, Analog Code andUniform Shape

As described above, analog-encoded microcarriers are highly advantageousfor multiplexed assays due to the vast number of potential uniqueidentifiers and reduced recognition error. This Example describesvarious types of microcarriers encoded with a two-dimensional shape,which may be used as an analog code for identification. It is to beunderstood that the encoded microcarriers of the present disclosure mayinclude some or all of the optional features set forth below in anycombination.

FIGS. 1A & 1B show two views of exemplary microcarrier 100. Microcarrier100 is a circular disc of approximately 50 μm in diameter and 10 μm inthickness. FIG. 1A provides a view of microcarrier 100 looking at acircular face of the disc, while FIG. 1B shows a side view ofmicrocarrier 100 orthogonal to the surface shown in FIG. 1A. Twocomponents of microcarrier 100 are shown. First, substantiallytransparent polymer layer 102 provides the body of the microcarrier.Layer 102 may be produced, e.g., using a polymer such as SU-8, asdescribed above.

Substantially non-transparent polymer layer 104 is affixed to a surfaceof layer 102. While the cross-section of microcarrier 100 shown in FIG.1B shows a discontinuous view of layer 104, the view shown in FIG. 1Aillustrates that layer 104 is shaped like a circular gear with aplurality of teeth. The shape, number, size, and spacing of these gearteeth constitutes a two-dimensional shape, and one or more of theseaspects of the gear teeth may be modified in order to produce multipletwo-dimensional shapes for analog encoding. Advantageously, the outsideedge of layer 104's gear teeth fit within the perimeter of layer 102.This allows for a variety of analog codes, each representing a uniqueidentifier for one species of microcarrier, while maintaining a uniformoverall shape across multiple species of microcarrier. Stated anotherway, each microcarrier species within a population of multiple speciesmay have a different two-dimensional gear shape (i.e., analog code), buteach microcarrier will have the same perimeter, leading to greateruniformity of physical properties (e.g., size, shape, behavior insolution, and the like). Layer 104 may be produced, e.g., using apolymer such as SU-8 mixed with a dye, or using a black matrix resist,as described above.

Layer 104 surrounds center portion 106 of layer 102. A capture agent forcapturing an analyte is coupled to at least center portion 106 on one orboth surfaces (i.e., upper/lower surfaces) of layer 102. Advantageously,this allows center portion 106 to be imaged without any potential forinterference resulting from layer 104.

FIGS. 1C & 1D show an exemplary assay using microcarrier 100 for analytedetection. FIG. 1C shows that microcarrier 100 may include capture agent108 coupled to one or more surfaces in at least center portion 106.Microcarrier 100 is contacted with a solution containing analyte 110,which is captured by capture agent 108. As described above, variouscapture agents may be used to capture different types of analytes,ranging from small molecules, nucleic acids, and proteins (e.g.,antibodies) to organelles, viruses, and cells. FIG. 1C illustrates asingle microcarrier species (i.e., microcarrier 100), which capturesanalyte 110, but in a multiplex assay multiple microcarrier species areused, each species having a particular capture agent that recognizes aspecific analyte.

FIG. 1D illustrates an exemplary process for “reading” microcarrier 100.This process includes two steps that may be accomplished simultaneouslyor separately. First, the capture of analyte 110 by capture agent 108 isdetected. In the example shown in FIG. 1D, detection agent 114 binds toanalyte 110. Analyte not captured by a capture agent coupled tomicrocarrier 100 may have been washed off prior to detection, such thatonly analytes bound to microcarrier 100 are detected. Detection agent114 also includes a reagent for detection. As one example, detectionagent 114 may include a fluorophore that, when excited by light 116 at awavelength within the excitation spectrum of the fluorophore, emitslight 118 (e.g., a photon). Light 118 may be detected by any suitabledetection means, such as a fluorescence microscope, plate reader, andthe like.

In addition, microcarrier 100 is read for its unique identifier. In theexample shown in FIG. 1D, light 112 is used to illuminate the fieldcontaining microcarrier 100 (in some embodiments, light 112 may have adifferent wavelength than lights 116 and 118). When light 112illuminates the field containing microcarrier 100, it passes throughsubstantially transparent polymer layer 102 but is blocked bysubstantially non-transparent polymer layer 104, as shown in FIG. 1D.This generates a light pattern that can be imaged, for example, by lightmicroscopy (e.g., using differential interference contrast, or DIC,microscopy). This light pattern is based on the two-dimensional shape(i.e., analog code) of microcarrier 100. Standard image recognitiontechniques may be used to decode the analog code represented by theimage of microcarrier 100.

The analyte detection and identifier imaging steps may occur in anyorder, or simultaneously. Advantageously, both detection steps shown inFIG. 1D may be accomplished on one imaging device. As one example, amicroscope capable of both fluorescence and light (e.g., bright field)microscopy may be used to quantify the amount of analyte 110 bound tomicrocarrier 100 (e.g., as detected by detection agent 114) and imagethe analog code created by layers 102 and 104. This allows for a moreefficient assay process with fewer equipment requirements.

Turning now to FIGS. 2A & 2B, another exemplary microcarrier 200 isshown. Like microcarrier 100, microcarrier 200 includes substantiallytransparent polymer layer 202 and substantially non-transparent polymerlayer 204. In addition, microcarrier 200 includes magnetic layer 206. Asshown in FIG. 2A, magnetic layer 206 may be shaped as a ring betweencenter portion 208 and substantially non-transparent layer 204.

FIG. 2B shows that magnetic layer 206 may be embedded within layer 202.Layer 202 may also include more than one layer, such that magnetic layer206 is sandwiched between two substantially transparent polymer layers(e.g., as in FIG. 2B). Alternatively, magnetic layer 206 may be affixedto the same surface of layer 202 as layer 204, or magnetic layer 206 maybe affixed to the surface of layer 202 opposite layer 204. In someembodiments, magnetic layer 206 may include nickel.

Magnetic layer 206 bestows magnetic properties onto microcarrier 200,which advantageously may be used for many applications. For example,microcarrier 200 may be affixed to a surface by magnetic attractionduring a washing step, allowing for effective washing without losing orotherwise disrupting the microcarriers.

In addition to its magnetic properties, layer 206 is also substantiallynon-transparent. When imaged as shown in FIG. 1D (e.g., using light112), layer 206 will block, either in part or in whole, transmittedlight, thereby creating a pattern for imaging. As shown in FIG. 2A,layer 206 is also asymmetric—in this example, it includes gap 210. Thisasymmetry creates an orientation indicator that can be imaged, forexample, as shown in FIG. 1D using light 112. Advantageously, anorientation indicator may be utilized during image recognition to orientthe two-dimensional shape created by imaging layer 204 in a uniformorientation for easier analog code recognition. This allowsmicrocarriers imaged in any orientation to be decoded.

FIG. 3 shows the vast number of potential analog codes possible usingthe gear shape shown in FIGS. 1A-2B. FIG. 3 illustrates an exemplarycoding scheme in which multiple shape variation points are labeled,e.g., at positions 302, 304, 306, 308, 310, 312, 314, 316, 318, 320,322, 324, 326, and 328 on exemplary microcarrier 300. Even if a simple“filled or not filled” scheme is used, up to 2¹⁴ unique codes arepossible based on the use of 14 shape variation points. This scheme isconvenient for both manufacturing and for generating two-dimensionalshapes that are easily distinguishable for image recognition analysis.However, since analog encoding is used, more complex schemes using morethan 2 possibilities (e.g., at each shape variation point as labeled inFIG. 3 ) are possible, thereby exponentially expanding the number ofunique identifiers. For example, multiple gear tooth shapes and/ormultiple sizes of gear teeth are possible. A two-dimensional gear shapeas shown in FIGS. 1A-3 facilitates a wide range of unique analog codeswhile providing a large center portion (e.g., center portions 106 and208) for analyte detection.

FIG. 4A illustrates three exemplary embodiments of the coding schemeshown in FIG. 3 : microcarriers 400, 402, and 404. The unique codes ofmicrocarriers 400, 402, and 404 are generated using the simple “filledor not filled” scheme of FIG. 3 . FIG. 4B illustrates 10 exemplaryembodiments of the cod, inter alia, in terms of number of shapes (e.g.,two distinct shapes in code ZN_3, as compared to seven distinct shapesin code ZN_10) and/or size of shapes (e.g., large, small, andintermediate-sized shapes in code ZN_2). Importantly, as describedabove, more complex encoding schemes are available using analog imagerecognition, thereby greatly expanding the number of potential uniquecodes.

Turning now to FIGS. 5A & 5B, another exemplary microcarrier 500 isshown. Like microcarrier 200, microcarrier 500 includes substantiallytransparent polymer layer 502, substantially non-transparent polymerlayer 504, magnetic layer 506, and center portion 508. In addition,microcarrier 500 has four columns including column 510, which may be ofany shape that extends from the surface of layer 502. As shown in FIG.5A, these columns may be arrayed in alignment with magnetic layer 506,preventing any potential for interfering with analyte detection incenter portion 508 or with reading the two-dimensional shape (i.e., theanalog code) of layer 504. FIG. 5B shows that these columns may extendfrom the upper and lower surfaces of microcarrier 500. Column 510 may bemade, for example, using the same substantially transparent polymer aslayer 502 (exemplary methods of production are described infra).Advantageously, one or more columns such as column 510 may be used toprevent microcarriers from sticking to each other and/or a container(e.g., the side of a well in a multiwell plate), e.g., through opticalcontact bonding.

Example 2 Microcarriers with a Two-Dimensional, Analog Code Encoded inthe Microcarrier Shape

The previous Example illustrates multiple exemplary embodiments ofmicrocarriers in which an analog code is provided by a non-transparentlayer affixed to a transparent polymer layer. This is advantageous, forexample, in allowing greater uniformity between different species ofmicrocarriers (i.e., each has the same perimeter shape provided by thetransparent polymer layer).

However, it may be advantageous for other reasons to use the perimeterof the microcarrier itself as the two-dimensional shape for analogencoding. For example, if the analog code is provided by the shape ofthe microcarrier itself, only one layer is required, therebystreamlining the manufacturing process. Moreover, shaping the perimeterof the microcarrier may be accomplished by highly precise manufacturingtechniques, allowing a highly reproducible shape for more accurate imagerecognition.

FIGS. 6A & 6B show exemplary microcarrier 600 of this type. Microcarrier600 is a gear-shaped disc approximately 80 μm in diameter and 15 μm inheight, including optional column elements (similar to column 510 asdescribed above). Microcarrier 600 is made of a single, non-transparentpolymer layer 602, rather than separate transparent and non-transparentpolymer layers. Microcarrier 600 may be imaged as shown in FIG. 1D, butits analog code is imaged based on the entire microcarrier shape (e.g.,perimeter of the non-transparent polymer layer). One or both surfaces ofmicrocarrier 600 may be used for coupling a capture agent as above, anda center portion or the entire surface may be used.

FIG. 6C illustrates the dimensions of gear tooth 604 of microcarrier600. As shown, in this embodiment, gear tooth 604 is 4 μm wide andspaced 4 μm from adjacent gear tooth 606. Since the two-dimensionalshape of microcarrier 600 is analog encoded, the perimeter betweenadjacent gear teeth may be variable, allowing for multiple gear toothshapes. For example, gear tooth 604 extends 4 or 6.5 μm in height,relative to the adjacent perimeter segment immediately to the left orright, respectively.

FIG. 7 illustrates another embodiment of this type of microcarrier,microcarrier 700. Like microcarrier 600, microcarrier 700 is made fromnon-transparent polymer layer 702. In addition, microcarrier includesmagnetic layer 704. Magnetic layer 704 may be affixed to one of thesurfaces of microcarrier 700, or it may be embedded within microcarrier700 (e.g., between two non-transparent polymer layers). Magnetic layer704 may be generated, for example, by depositing nickel. As describedabove, a magnetic layer allows additional functionalities, such as theoption for washing microcarrier 700 while magnetically attached toanother surface.

Turning now to FIG. 8A, another exemplary microcarrier 800 is shown.Like microcarrier 700, microcarrier 800 includes non-transparent polymerlayer 802 (and optionally, a magnetic layer such as layer 704). Inaddition, microcarrier 800 includes start position 804, which has adifferent shape than the rest of the perimeter of microcarrier 800.Start position 804 may be used as an orientation indicator for imagerecognition, as described above in reference to gap 210 shown in FIG.2A.

FIG. 8B illustrates a coding scheme that may be used. FIG. 8B showsmicrocarrier 810, which like microcarrier 800 includes non-transparentpolymer layer 812 and start position 814 (and optionally, a magneticlayer such as layer 704). In this scheme, potential shape variationpoints around the gear are labeled, e.g., at positions 820, 822, 824,826, 828, 830, 832, 834, 836, 838, 840, 842, and 844. As shown in FIG.8B, even if only two potential shapes may be used for positions 820,822, 824, 826, 828, 830, 832, 834, 836, 838, 840, 842, and 844, thisembodiment allows up to 2¹³ unique codes. Further, as described above,the use of analog encoding greatly expands this number by allowing theuse of more than two potential shapes at any or all of the indicatedpositions around the perimeter (e.g., at each shape variation point aslabeled in FIG. 8B).

FIGS. 9A-9C illustrate yet another potential embodiment in microcarrier900. Like microcarrier 800, microcarrier 900 is a gear-shapedmicrocarrier that includes non-transparent polymer layer 902 and startposition 904 (and optionally, a magnetic layer such as layer 704). Inaddition, microcarrier 900 may have one or more columns (e.g., column906) affixed to one or both surfaces of microcarrier 900. As shown inthe cross-section in FIG. 9B, column 906 extends from a surface of layer902. Advantageously, column 906 helps to reduce the potential foroptical contact bonding (as described above in reference to column 510).

FIG. 9C illustrates the dimensions of column 906. In this example,column 906 is a cylinder 3 μm in height and 3 μm in diameter, althoughas described above such columns are in no way limited to a cylindricalshape. In some embodiments, column 906 is made of a magnetic material,such as nickel. This allows column 906 to function additionally as amagnetic element for magnetic manipulation of microcarrier 900, asdescribed above.

Example 3 Methods of Producing Microcarriers with a Two-Dimensional,Analog Code Encoded in the Microcarrier Shape

Having described exemplary embodiments of multiple types ofmicrocarriers in the previous Examples, attention is now directed tomethods of producing microcarriers. As described above, themicrocarriers of the present disclosure may be made of one, two, or moreconstituent layers, depending on the desired configuration and/oroptional features.

Process 1000 shown in FIG. 10 illustrates an exemplary workflow formanufacturing a single layer microcarrier, such as those described inExample 2 above. At block 1002, sacrificial layer 1006 is constructed onsubstrate 1004. In some embodiments, substrate 1004 may be a glasssubstrate. At block 1010, layer 1012 is deposited on sacrificial layer1006. In some embodiments, layer 1012 is a non-transparent polymerlayer. At block 1020, the perimeter of layer 1012 is shaped into a gearshape (as described above) using lithography to generate gear-shapedlayer 1022. At block 1030, the entire structure (i.e., layer 1022,sacrificial layer 1006, and substrate 1004) is immersed in a solvent.This solvent treatment dissolves sacrificial layer 1006 and releasesgear-shaped layer 1022 from substrate 1004, thereby generatingmicrocarrier 1032. In some embodiments, microcarrier 1032 may be furthermodified, for example, by coupling a capture agent to one or bothsurfaces.

As described in Example 2 above, gear-shaped microcarriers may includeoptional elements such as magnetic components (e.g., columns and/ormagnetic layers). Process 1100 shown in FIGS. 11A & 11B illustrates anexemplary workflow for manufacturing gear-shaped microcarriers with oneor more magnetic components.

As shown in FIG. 11A, at block 1102, sacrificial layer 1106 isconstructed on substrate 1104. In some embodiments, substrate 1104 maybe a glass substrate. At block 1110, magnetic layer 1112 is deposited onsacrificial layer 1106. In some embodiments, magnetic layer 1112includes nickel. At block 1120, magnetic layer 1112 is shaped bylithography into shaped magnetic layer 1122. Shaped magnetic layer 1122may take any desired shape, e.g., it may be shaped into one or morecolumns, as illustrated in FIG. 9A with column 906.

As shown in FIG. 11B, at block 1130, substantially non-transparentpolymer layer 1132 is deposited over shaped magnetic layer 1122 andsacrificial layer 1106. At block 1140, the perimeter of layer 1132 isshaped by lithography into gear-shaped substantially non-transparentlayer 1142 (such as one of the gear shapes illustrated in FIGS. 6A-9A).At block 1150, the entire structure (i.e., layer 1142, shaped magneticlayer 1122, sacrificial layer 1106, and substrate 1104) is immersed in asolvent. This solvent treatment dissolves sacrificial layer 1106 andreleases gear-shaped layer 1142 and shaped magnetic layer 1122 fromsubstrate 1104, thereby generating microcarrier 1152. In someembodiments, microcarrier 1152 may be further modified, for example, bycoupling a capture agent to one or both surfaces.

Example 4 Methods of Producing Encoded Microcarriers with aTwo-Dimensional, Analog Code and Uniform Shape

Attention is now directed to methods of producing encoded microcarrierswith a one or more substantially transparent and one or moresubstantially non-transparent polymer layers, such as those described inExample 1. FIGS. 12A-12E illustrate process 1200, an exemplary workflowfor manufacturing microcarriers with a substantially transparent polymerlayer, a substantially non-transparent polymer layer (whosetwo-dimensional shape constitutes an analog code), and one or morecolumns.

Beginning with FIG. 12A, at block 1202, sacrificial layer 1206 isdeposited (e.g., by spin-coating) onto substrate 1204. In someembodiments, substrate 1204 may be a glass substrate. At block 1208,mask 1210 is applied, and sacrificial layer 1206 is exposed with UVlight. UV light is applied through mask 1210, allowing UV light segments1212 and 1214 to pass through and treat sacrificial layer 1206. At block1216, after development of the structure through standard lithographicdevelopment, sacrificial layer 1206 is shaped into shaped sacrificiallayer 1218 as a result of the masking of the UV treatment.

Process 1200 continues at block 1220 (FIG. 12B), where the masked holesin shaped sacrificial layer 1218 are filled with a substantiallytransparent polymer, creating columns 1222 and 1224. At block 1226,substantially transparent polymer layer 1228 is deposited over columns1222 and 1224, as well as shaped sacrificial layer 1218.

Process 1200 continues at block 1230 (FIG. 12C), where magnetic layer1232 is deposited over layer 1228. In some embodiments, magnetic layer1232 includes nickel. In some embodiments, magnetic layer 1232 isdeposited by sputtering. At block 1234, an etch-block layer is depositedover magnetic layer 1232, as represented by etch-blocks 1236 and 1238.At block 1240, the unblocked segments of magnetic layer 1232 are etchedout, generated shaped magnetic layer 1242. In some embodiments, shapedmagnetic layer 1242 may be shaped into a ring shape (with optionalasymmetry for indication of orientation) surrounding a center portion oflayer 1228 (see, e.g., layer 206 in FIG. 2A). At block 1244, theetch-block layer (as represented by etch-blocks 1236 and 1238) isremoved.

Process 1200 continues at block 1246 (FIG. 12D), where substantiallytransparent polymer layer 1248 is deposited over layers 1228 and 1242(filling in any holes in layer 1242 created by etch-blocking). At block1250, substantially non-transparent layer 1252 is deposited and shapedby lithography on top of layer 1248. In some embodiments, layer 1252 isshaped with one or more gear teeth in a ring surrounding magnetic layer1242 (see, e.g., layer 204 in relation to layers 202 and 206 and centerportion 208 of FIG. 2A).

Process 1200 continues at block 1254 (FIG. 12E), where columns 1256 and1258 are shaped by lithography on top of layer 1248. In someembodiments, columns 1256 and 1258 are made of a substantiallytransparent polymer. In some embodiments, the columns are positioned asshown in FIGS. 5A & 5B. At block 1260, substrate 1204 is cut into one ormore microcarriers of the same shape (i.e., although for simplicity ofexplanation only one microcarrier is depicted in FIGS. 12A-12E, morethan 1 microcarrier may be constructed on substrate 1204 in process1200). Also at block 1260, the entire structure (i.e., including 1204,1218, 1222, 1224, 1228, 1242, 1248, 1252, 1256, and 1258) is immersed ina solvent. This solvent treatment dissolves sacrificial layer 1218 andreleases microcarrier 1262 from substrate 1204. In some embodiments,microcarrier 1262 may be further modified, for example, by coupling acapture agent to one or both surfaces.

FIGS. 13A-13C illustrate process 1300, an exemplary workflow forgenerating a different type of multi-layer microcarrier. Beginning withFIG. 13A, at block 1302, sacrificial layer 1306 is deposited onsubstrate layer 1304. In some embodiments, substrate 1304 is a glasssubstrate. At block 1308, substantially transparent layer 1310 isdeposited over sacrificial layer 1306. At block 1312, magnetic layer1314 is deposited over layer 1310. In some embodiments, magnetic layer1314 includes nickel.

Process 1300 continues at block 1316 (FIG. 13B), where magnetic layer1314 is defined into shaped magnetic layer 1318. In some embodiments,shaped magnetic layer 1318 is defined into a ring shape (with optionalasymmetry for indication of orientation) surrounding a center portion oflayer 1310 (see, e.g., layer 206 in FIG. 2A). At block 1320,substantially transparent layer 1322 is deposited over layers 1318 and1310, filling in any holes created by defining shaped layer 1318. Atblock 1324, substantially non-transparent polymer layer 1326 isdeposited over layer 1322.

Process 1300 continues at block 1328 (FIG. 13C), where substantiallynon-transparent polymer layer 1326 is shaped by lithography intogear-shaped substantially non-transparent polymer layer 1330. In someembodiments, layer 1330 is shaped with one or more gear teeth in a ringsurrounding shaped magnetic layer 1318 (see, e.g., layer 204 in relationto layers 202 and 206 and center portion 208 of FIG. 2A). At block 1332,the entire structure (i.e., including 1304, 1306, 1310, 1318, 1322, and1330) is immersed in a solvent. This solvent treatment dissolvessacrificial layer 1306 and releases microcarrier 1334 from substrate1304. In some embodiments, microcarrier 1334 may be further modified,for example, by coupling a capture agent to one or both surfaces.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

What is claimed is:
 1. An encoded microcarrier, comprising: (a) asubstantially transparent polymer layer having a first surface and asecond surface, the first and the second surfaces being parallel to eachother; (b) a substantially non-transparent polymer layer, wherein thesubstantially non-transparent polymer layer is a discontinuous,substantially non-transparent polymer layer affixed to the first surfaceof the substantially transparent polymer layer that encloses a centerportion of the substantially transparent polymer layer, wherein thediscontinuous, substantially non-transparent polymer layer represents ananalog code; and (c) a capture agent for capturing an analyte, whereinthe capture agent is coupled to at least one of the first surface andthe second surface of the substantially transparent polymer layer in atleast the center portion of the substantially transparent polymer layer.2. The microcarrier of claim 1, further comprising: (d) a magnetic,substantially non-transparent layer that encloses the center portion ofthe substantially transparent polymer layer between the substantiallynon-transparent polymer layer and the center portion of thesubstantially transparent polymer layer, wherein the magnetic,substantially non-transparent layer is affixed to the first surface orthe second surface of the substantially transparent polymer layer. 3.The microcarrier of claim 1, further comprising: (e) a secondsubstantially transparent polymer layer aligned with the firstsubstantially transparent polymer layer, the second substantiallytransparent polymer layer having a center portion that is aligned withthe center portion of the first substantially transparent polymer layer,wherein the second substantially transparent polymer layer is affixed tothe second surface of the first substantially transparent polymer layerand does not extend beyond the two-dimensional shape of the firstsubstantially transparent polymer layer; and (f) a magnetic,substantially non-transparent layer that encloses the center portion ofthe first substantially transparent polymer layer between thesubstantially non-transparent polymer layer and the center portion ofthe substantially transparent polymer layer, wherein the magnetic,substantially non-transparent layer is affixed between the first and thesecond substantially transparent polymer layers.
 4. The microcarrier ofclaim 1, further comprising an orientation indicator for orienting theanalog code of the substantially non-transparent polymer layer.
 5. Themicrocarrier of claim 4, wherein the orientation indicator comprises anasymmetry of the magnetic, substantially non-transparent layer.
 6. Themicrocarrier of claim 2, wherein the magnetic, substantiallynon-transparent layer comprises nickel.
 7. The microcarrier of claim 2,wherein the magnetic, substantially non-transparent layer is betweenabout 50 nm and about 10 μm in thickness.
 8. The microcarrier of claim7, wherein the magnetic, substantially non-transparent layer is about0.1 μm in thickness.
 9. The microcarrier of claim 1, wherein thediscontinuous, substantially non-transparent polymer layer comprises agear shape comprising a plurality of gear teeth, and wherein the analogcode is represented by one or more aspects selected from the groupconsisting of the height of one or more gear teeth of the plurality, thewidth of one or more gear teeth of the plurality, the number of gearteeth in the plurality, and the arrangement of one or more gear teethwithin the plurality.
 10. The microcarrier of claim 1, furthercomprising: (g) one or more columns projecting from the first surface ofthe first substantially transparent polymer layer, wherein the one ormore columns are not within the center portion of the firstsubstantially transparent polymer layer; and/or (h) one or more columnsprojecting from the second surface of the first substantiallytransparent polymer layer or a surface of the second substantiallytransparent polymer layer that is not affixed to the first substantiallytransparent polymer layer, wherein the one or more columns are notwithin the center portions of the first or the second substantiallytransparent polymer layer.
 11. The microcarrier of claim 1, wherein themicrocarrier is a substantially circular disc.
 12. The microcarrier ofclaim 1, wherein the center portion of the first substantiallytransparent polymer layer comprises between about 5% and about 90% ofthe surface area of the first substantially transparent polymer layer.13. The microcarrier of claim 12, wherein the center portion of thefirst substantially transparent polymer layer comprises about 25% of thesurface area of the first substantially transparent polymer layer. 14.The microcarrier of claim 1, wherein the microcarrier is less than about200 μm in diameter.
 15. The microcarrier of claim 14, wherein themicrocarrier is about 50 μm in diameter.
 16. The microcarrier of claim1, wherein the microcarrier is less than about 50 μm in thickness. 17.The microcarrier of claim 16, wherein the microcarrier is about 10 μm inthickness.
 18. The microcarrier of claim 1, wherein the analyte isselected from the group consisting of a DNA molecule, aDNA-analog-molecule, an RNA-molecule, an RNA-analog-molecule, apolynucleotide, a protein, an enzyme, a lipid, a phospholipid, acarbohydrate moiety, a polysaccharide, an antigen, a virus, a cell, anantibody, a small molecule, a bacterial cell, a cellular organelle, andan antibody fragment.
 19. The microcarrier of claim 1, wherein thecapture agent for capturing the analyte is selected from the groupconsisting of a DNA molecule, a DNA-analog-molecule, an RNA-molecule, anRNA-analog-molecule, a polynucleotide, a protein, an enzyme, a lipid, aphospholipid, a carbohydrate moiety, a polysaccharide, an antigen, avirus, a cell, an antibody, a small molecule, a bacterial cell, acellular organelle, and an antibody fragment.
 20. The microcarrier ofclaim 1, wherein the substantially transparent polymer of the first orthe second substantially transparent polymer layer comprises anepoxy-based polymer.
 21. The microcarrier of claim 20, wherein theepoxy-based polymer is SU-8.
 22. A plurality of encoded microcarriers,wherein each encoded microcarrier of the plurality comprises: (a) asubstantially transparent polymer layer having a first surface and asecond surface, the first and the second surfaces being parallel to eachother; (b) a substantially non-transparent polymer layer, wherein thesubstantially non-transparent polymer layer is a discontinuous,substantially non-transparent polymer layer affixed to the first surfaceof the substantially transparent polymer layer that encloses a centerportion of the substantially transparent polymer layer, wherein thediscontinuous, substantially non-transparent polymer layer represents ananalog code; and (c) a capture agent for capturing an analyte, whereinthe capture agent is coupled to at least one of the first surface andthe second surface of the substantially transparent polymer layer in atleast the center portion of the substantially transparent polymer layer.23. The plurality of encoded microcarriers of claim 22, comprising afirst and a second species of microcarriers; wherein the discontinuous,substantially non-transparent polymer layer of the microcarriers of thefirst species represents a first analog code, and the microcarriers ofthe first species comprise a first capture agent for capturing a firstanalyte; wherein the discontinuous, substantially non-transparentpolymer layer of the microcarriers of the second species represents asecond analog code, and the microcarriers of the second species comprisea second capture agent for capturing a second analyte; and wherein thefirst and second analog codes are different.
 24. The plurality ofencoded microcarriers of claim 23, wherein the first and second captureagents are different.
 25. The plurality of encoded microcarriers ofclaim 24, wherein the first and second analytes are different.